Unsolvated benzodiazepine compositions and methods

ABSTRACT

The present invention relates to systems and methods for generating new forms of benzodiazepine and benzodiazepine related compounds as well as new compounds and formulations generated by such methods. In particular, the present invention provides high throughput systems and methods for generating and identifying new crystalline benzodiazepine and benzodiazepine related polymorphs and new unsolvated, solvated, and other forms of the compounds that find use as improved drugs and drug formations.

CROSS-REFERENCE TO RELATED APPLICATIONS

This application is a continuation of pending U.S. patent applicationSer. No. 11/445,010, filed Jun. 1, 2006, which claims priority toexpired U.S. Provisional Application Ser. Nos. 60/686,348, filed Jun. 1,2005, and 60/704,102, filed Jul. 29, 2005, the disclosures of which areherein incorporated by reference in its entirety.

STATEMENT REGARDING FEDERALLY SPONSORED RESEARCH OR DEVELOPMENT

This invention was made with government support under Grant No. GM046831awarded by the National Institutes of Health. The government has certainrights in the invention.

FIELD OF THE INVENTION

The present invention relates to systems and methods for generating newforms of benzodiazepine and benzodiazepine related compounds as well asnew compounds and formulations generated by such methods. In particular,the present invention provides high throughput systems and methods forgenerating and identifying new crystalline benzodiazepine andbenzodiazepine related polymorphs and new unsolvated, solvated, andother forms of the compounds that find use as improved drugs and drugformations.

BACKGROUND OF THE INVENTION

Multicellular organisms exert precise control over cell number. Abalance between cell proliferation and cell death achieves thishomeostasis. Cell death occurs in nearly every type of vertebrate cellvia necrosis or through a suicidal form of cell death, known asapoptosis. Apoptosis is triggered by a variety of extracellular andintracellular signals that engage a common, genetically programmed deathmechanism.

Multicellular organisms use apoptosis to instruct damaged or unnecessarycells to destroy themselves for the good of the organism. Control of theapoptotic process therefore is very important to normal development, forexample, fetal development of fingers and toes requires the controlledremoval, by apoptosis, of excess interconnecting tissues, as does theformation of neural synapses within the brain. Similarly, controlledapoptosis is responsible for the sloughing off of the inner lining ofthe uterus (the endometrium) at the start of menstruation. Whileapoptosis plays an important role in tissue sculpting and normalcellular maintenance, it is also the primary defense against cells andinvaders (e.g., viruses) which threaten the well being of the organism.

Not surprisingly many diseases are associated with dysregulation of theprocess of cell death. Experimental models have established acause-effect relationship between aberrant apoptotic regulation and thepathenogenicity of various neoplastic, autoimmune and viral diseases.For instance, in the cell mediated immune response, effector cells(e.g., cytotoxic T lymphocytes “CTLs”) destroy virus-infected cells byinducing the infected cells to undergo apoptosis. The organismsubsequently relies on the apoptotic process to destroy the effectorcells when they are no longer needed. Autoimmunity is normally preventedby the CTLs inducing apoptosis in each other and even in themselves.Defects in this process are associated with a variety of autoimmunediseases such as lupus erythematosus and rheumatoid arthritis.

Multicellular organisms also use apoptosis to instruct cells withdamaged nucleic acids (e.g., DNA) to destroy themselves prior tobecoming cancerous. Some cancer-causing viruses overcome this safeguardby reprogramming infected (transformed) cells to abort the normalapoptotic process. For example, several human papilloma viruses (HPVs)have been implicated in causing cervical cancer by suppressing theapoptotic removal of transformed cells by producing a protein (E6) whichinactivates the p53 apoptosis promoter. Similarly, the Epstein-Barrvirus (EBV), the causative agent of mononucleosis and Burkitt'slymphoma, reprograms infected cells to produce proteins that preventnormal apoptotic removal of the aberrant cells thus allowing thecancerous cells to proliferate and to spread throughout the organism.

Still other viruses destructively manipulate a cell's apoptoticmachinery without directly resulting in the development of a cancer. Forexample, the destruction of the immune system in individuals infectedwith the human immunodeficiency virus (HIV) is thought to progressthrough infected CD4⁺ T cells (about 1 in 100,000) instructinguninfected sister cells to undergo apoptosis.

Some cancers that arise by non-viral means have also developedmechanisms to escape destruction by apoptosis. Melanoma cells, forinstance, avoid apoptosis by inhibiting the expression of the geneencoding Apaf-1. Other cancer cells, especially lung and colon cancercells, secrete high levels of soluble decoy molecules that inhibit theinitiation of CTL mediated clearance of aberrant cells. Faultyregulation of the apoptotic machinery has also been implicated invarious degenerative conditions and vascular diseases.

It is apparent that the controlled regulation of the apoptotic processand its cellular machinery is vital to the survival of multicellularorganisms. Typically, the biochemical changes that occur in a cellinstructed to undergo apoptosis occur in an orderly procession. However,as shown above, flawed regulation of apoptosis can cause seriousdeleterious effects in the organism.

There have been various attempts to control and restore regulation ofthe apoptotic machinery in aberrant cells (e.g., cancer cells). Forexample, much work has been done to develop cytotoxic agents to destroyaberrant cells before they proliferate. As such, cytotoxic agents havewidespread utility in both human and animal health and represent thefirst line of treatment for nearly all forms of cancer andhyperproliferative autoimmune disorders like lupus erythematosus andrheumatoid arthritis.

Many cytotoxic agents in clinical use exert their effect by damaging DNA(e.g., cis-diaminodichroplatanim(II) cross-links DNA, whereas bleomycininduces strand cleavage). The result of this nuclear damage, ifrecognized by cellular factors like the p53 system, is to initiate anapoptotic cascade leading to the death of the damaged cell.

However, existing cytotoxic chemotherapeutic agents have seriousdrawbacks. For example, many known cytotoxic agents show littlediscrimination between healthy and diseased cells. This lack ofspecificity often results in severe side effects that can limit efficacyand/or result in early mortality. Moreover, prolonged administration ofmany existing cytotoxic agents results in the expression of resistancegenes (e.g., bcl-2 family or multi-drug resistance (MDR) proteins) thatrender further dosing either less effective or useless. Some cytotoxicagents induce mutations into p53 and related proteins. Based on theseconsiderations, ideal cytotoxic drugs should only kill diseased cellsand not be susceptible to chemo-resistance.

One strategy to selectively kill diseased cells is to develop drugs thatselectively recognize molecules expressed in diseased cells. Thus,effective cytotoxic chemotherapeutic agents, would recognize diseaseindicative molecules and induce (e.g., either directly or indirectly)the death of the diseased cell. Although markers on some types of cancercells have been identified and targeted with therapeutic antibodies andsmall molecules, unique traits for diagnostic and therapeuticexploitation are not known for most cancers. Moreover, for diseases likelupus, specific molecular targets for drug development have not beenidentified.

What are needed are improved compositions and methods for regulating theapoptotic processes in subjects afflicted with diseases and conditionscharacterized by faulty regulation of these processes (e.g., viralinfections, hyperproliferative autoimmune disorders, chronicinflammatory conditions, and cancers).

SUMMARY

The present invention relates to systems and methods for generating newforms of benzodiazepine and benzodiazepine related compounds as well asnew compounds and formulations generated by such methods. In particular,the present invention provides high throughput systems and methods forgenerating and identifying new crystalline benzodiazepine andbenzodiazepine related polymorphs and new unsolvated and other forms ofthe compounds that find use as improved drugs and drug formations.

For example, the present invention provides unsolvated forms ofbenzodiazepine compounds and methods of making such compounds. In someembodiments, the benzodiazepine compounds have orthorhombic crystallineforms. The unsolvated benzodiazepines of the present invention find usein pharmaceutical formulations with enhanced properties (e.g.,shelf-life, tabletability, etc.). The present invention is illustratedwith the benzodiazepine, Bz-423. However, the present invention is notlimited to this particular compound. It will be understood that avariety of related compounds find use in the compositions and methods ofthe present invention.

In some embodiments, the benzodiazepines of the present invention haveorthorhombic crystals (e.g., Bz-423). In some preferred embodiments, thecompounds are anhydrous benzodiazepines, an ethanol solvate ofbenzodiazepines, a succinic acid (2:1) formulation of benzoediazepines,a citric acid (2:1) formulation of benzodiazepines, biphenyl derivateformulations of benzodiazepines, acetic acid formulations ofbenzodiazepine, CH₃CN formulations of benzodiazepine, methanolformulations of benzodiazepines, ethyl acetate formulations ofbenzodiazepines, toluene formulations of benzodiazepines, oxalic acidformulations of benzodiazepines, fumaric acid formulations ofbenzodiazepines, octanol formulations of benzodiazepines, heptanoic acidformulations of benzodiazepines, diphenyl ether formulations ofbenzodiazepines, and trichlorobenzene formulations of benzodiazepines.Other solvated, unsolvated and salt forms may also be used.

In certain embodiments, the present invention provides a compositioncomprising a solution of dissolved benzodiazepine (e.g., Bz-423) incontact with a crystal form (e.g., orthorhombic) of the samebenzodiazepine obtained from the solution, wherein the crystal form,when isolated, is capable of being provided in unsolvated form. Inpreferred embodiments, the composition further comprises a polymersurface in contact with the crystals. In preferred embodiments, thesolution comprises an aqueous solution.

In certain embodiments, the present invention provides a method forproducing orthorhombic Bz-423 crystals comprising providing the abovedescribed composition and isolating the crystals.

In preferred embodiments, the method further comprises the step ofpreparing benzodiazepines to generated the above described compositions.

The present invention further provides methods of preparing apharmaceutical preparation comprising new benzodiazepine compositions(alone, or in combination with other drugs or agents). In preferredembodiments, the pharmaceutical preparation comprises a tablet.

In preferred embodiments, the method further comprises the step ofselling the pharmaceutical preparation, prescribing the pharmaceuticalpreparation to a patient, and/or administering the pharmaceuticalpreparation to a subject (e.g., human).

In certain embodiments, the present invention provides a method forproducing a crystal form of a benzodiazepine described above comprisingexposing a solution containing a benzodiazepine to a polymer underconditions that permit crystal formation.

In certain embodiments, the present invention provides a method oftreating an autoimmune disorder or hyperproliferative disordercomprising administering to a subject an effective amount of acomposition comprising the new benzodiazepine formulations describedabove.

In preferred embodiments, the composition comprises an oral dose of thenew benzodiazepine formulations described above.

In certain embodiments, the present invention provides a compositioncomprising an unsolvated compound having the structure described by thefollowing formula:

including both R and S enantiomeric foms and racemic mixtures; whereinR1, R2, R3 and R4 are selected from the group consisting of: hydrogen;CH₃; a linear or branched, saturated or unsaturated aliphatic chainhaving at least 1 carbon; a linear or branched, saturated or unsaturatedaliphatic chain having at least 2 carbons, and having at least onehydroxy subgroup; a linear or branched, saturated or unsaturatedaliphatic chain having at least 2 carbons, and having at least one thiolsubgroup; a linear or branched, saturated or unsaturated aliphatic chainhaving at least 2 carbons, wherein the aliphatic chain terminates withan aldehyde subgroup; a linear or branched, saturated or unsaturatedaliphatic chain having at least 2 carbons, and having at least oneketone subgroup; a linear or branched, saturated or unsaturatedaliphatic chain having at least 2 carbons; wherein the aliphatic chainterminates with a carboxylic acid subgroup; a linear or branched,saturated or unsaturated aliphatic chain having at least 2 carbons, andhaving at least one amide subgroup; a linear or branched, saturated orunsaturated aliphatic chain having at least 2 carbons, and having atleast one acyl group; a linear or branched, saturated or unsaturatedaliphatic chain having at least 2 carbons, and having at least onenitrogen containing moiety; a linear or branched, saturated orunsaturated aliphatic chain having at least 2 carbons, and having atleast one amine subgroup; a linear or branched, saturated or unsaturatedaliphatic chain having at least 2 carbons, and having at least one ethersubgroup; a linear or branched, saturated or unsaturated aliphatic chainhaving at least 2 carbons, and having at least one halogen subgroup; alinear or branched, saturated or unsaturated aliphatic chain having atleast 2 carbons, and having at least one nitronium subgroup; wherein R5is selected from the group consisting of: OH; NO₂; OR′; wherein R′ isselected from the group consisting of: a linear or branched, saturatedor unsaturated aliphatic chain having at least one carbon; a linear orbranched, saturated or unsaturated aliphatic chain having at least 2carbons, and having at least one hydroxyl subgroup; a linear orbranched, saturated or unsaturated aliphatic chain having at least 2carbons, and having at least one thiol subgroup; a linear or branched,saturated or unsaturated aliphatic chain having at least 2 carbons,wherein the aliphatic chain terminates with an aldehyde subgroup; alinear or branched, saturated or unsaturated aliphatic chain having atleast 2 carbons, and having at least one ketone subgroup; a linear orbranched, saturated or unsaturated aliphatic chain having at least 2carbons; wherein the aliphatic chain terminates with a carboxylic acidsubgroup; a linear or branched, saturated or unsaturated aliphatic chainhaving at least 2 carbons, and having at least one amide subgroup; alinear or branched, saturated or unsaturated aliphatic chain having atleast 2 carbons, and having at least one acyl group; a linear orbranched, saturated or unsaturated aliphatic chain having at least 2carbons, and having at least one nitrogen containing moiety; a linear orbranched, saturated or unsaturated aliphatic chain having at least 2carbons, and having at least one amine subgroup; a linear or branched,saturated or unsaturated aliphatic chain having at least 2 carbons, andhaving at least one halogen subgroup; a linear or branched, saturated orunsaturated aliphatic chain having at least 2 carbons, and having atleast one nitronium subgroup; wherein R6 is selected from the groupconsisting of: Hydrogen; NO₂; Cl; F; Br; I; SR′; and NR′₂; wherein R′ isdefined as above in R5;wherein R7 is selected from the group consistingof: Hydrogen; a linear or branched, saturated or unsaturated aliphaticchain having at least 2 carbons; and wherein R8 is an aliphatic cyclicgroup larger than benzene; wherein the larger than benzene comprises anychemical group containing 7 or more non-hydrogen atoms.

In preferred embodiments, the compound is:

In preferred embodiments, the unsolvated compound is anhydrous. Inpreferred embodiment, the unsolvated compound has an orthorhombiccrystal structure.

In certain embodiments, the present invention provides a compositioncomprising a compound selected from the group consisting of Bz-423ethanol solvate, Bz-423 succinic acid, Bz-423 citric acid, Bz-423biphenyl derivate, BZ-423-acetic acid, BZ-423-CH₃ CN, BZ-423-methanol,BZ-423-ethyl acetate, BZ-423-toluene, BZ-423-oxalic acid, BZ-423-fumaricacid, BZ-423-octanol, BZ-423-heptanoic acid, BZ-423-diphenyl ether,Bz-423 1-propanol solvate, Bz-423 2-propanol solvate, Bz-423 1-butanolsolvate, Bz-423 2-butanol solvate, Bz-423 1-pentanol solvate, Bz-423propylene glycol, , Bz-423 1-octanol solvate, Bz-423 acetone glass, andBZ-423-trichlorobenzene.

In certain embodiments, the present invention provides a compositioncomprising an orthorhombic benzodiazepine crystal, the benzodiazepinehaving the structure:

including both R and S enantiomeric foms and racemic mixtures; whereinR1, R2, R3 and R4 are selected from the group consisting of: hydrogen;CH₃; a linear or branched, saturated or unsaturated aliphatic chainhaving at least 1 carbon; a linear or branched, saturated or unsaturatedaliphatic chain having at least 2 carbons, and having at least onehydroxy subgroup; a linear or branched, saturated or unsaturatedaliphatic chain having at least 2 carbons, and having at least one thiolsubgroup; a linear or branched, saturated or unsaturated aliphatic chainhaving at least 2 carbons, wherein the aliphatic chain terminates withan aldehyde subgroup; a linear or branched, saturated or unsaturatedaliphatic chain having at least 2 carbons, and having at least oneketone subgroup; a linear or branched, saturated or unsaturatedaliphatic chain having at least 2 carbons; wherein the aliphatic chainterminates with a carboxylic acid subgroup; a linear or branched,saturated or unsaturated aliphatic chain having at least 2 carbons, andhaving at least one amide subgroup; a linear or branched, saturated orunsaturated aliphatic chain having at least 2 carbons, and having atleast one acyl group; a linear or branched, saturated or unsaturatedaliphatic chain having at least 2 carbons, and having at least onenitrogen containing moiety; a linear or branched, saturated orunsaturated aliphatic chain having at least 2 carbons, and having atleast one amine subgroup; a linear or branched, saturated or unsaturatedaliphatic chain having at least 2 carbons, and having at least one ethersubgroup; a linear or branched, saturated or unsaturated aliphatic chainhaving at least 2 carbons, and having at least one halogen subgroup; alinear or branched, saturated or unsaturated aliphatic chain having atleast 2 carbons, and having at least one nitronium subgroup; wherein R5is selected from the group consisting of: OH; NO₂; OR′; wherein R′ isselected from the group consisting of: a linear or branched, saturatedor unsaturated aliphatic chain having at least one carbon; a linear orbranched, saturated or unsaturated aliphatic chain having at least 2carbons, and having at least one hydroxyl subgroup; a linear orbranched, saturated or unsaturated aliphatic chain having at least 2carbons, and having at least one thiol subgroup; a linear or branched,saturated or unsaturated aliphatic chain having at least 2 carbons,wherein the aliphatic chain terminates with an aldehyde subgroup; alinear or branched, saturated or unsaturated aliphatic chain having atleast 2 carbons, and having at least one ketone subgroup; a linear orbranched, saturated or unsaturated aliphatic chain having at least 2carbons; wherein the aliphatic chain terminates with a carboxylic acidsubgroup; a linear or branched, saturated or unsaturated aliphatic chainhaving at least 2 carbons, and having at least one amide subgroup; alinear or branched, saturated or unsaturated aliphatic chain having atleast 2 carbons, and having at least one acyl group; a linear orbranched, saturated or unsaturated aliphatic chain having at least 2carbons, and having at least one nitrogen containing moiety; a linear orbranched, saturated or unsaturated aliphatic chain having at least 2carbons, and having at least one amine subgroup; a linear or branched,saturated or unsaturated aliphatic chain having at least 2 carbons, andhaving at least one halogen subgroup; a linear or branched, saturated orunsaturated aliphatic chain having at least 2 carbons, and having atleast one nitronium subgroup; wherein R6 is selected from the groupconsisting of: Hydrogen; NO₂; Cl; F; Br; I; SR′; and NR′₂; wherein R′ isdefined as above in R5; wherein R7 is selected from the group consistingof: Hydrogen; a linear or branched, saturated or unsaturated aliphaticchain having at least 2 carbons; and wherein R8 is an aliphatic cyclicgroup larger than benzene; wherein the larger than benzene comprises anychemical group containing 7 or more non-hydrogen atoms.

In preferred embodiments, the compound is:

In preferred embodiments, the orthorhombic benzodiazepine crystal isanhydrous.

In certain embodiments, the present invention provides a compositioncomprising an oral dose of a benzodiazepine having the structure:

including both R and S enantiomeric foms and racemic mixtures; whereinR1, R2, R3 and R4 are selected from the group consisting of: hydrogen;CH₃; a linear or branched, saturated or unsaturated aliphatic chainhaving at least 1 carbon; a linear or branched, saturated or unsaturatedaliphatic chain having at least 2 carbons, and having at least onehydroxy subgroup; a linear or branched, saturated or unsaturatedaliphatic chain having at least 2 carbons, and having at least one thiolsubgroup; a linear or branched, saturated or unsaturated aliphatic chainhaving at least 2 carbons, wherein the aliphatic chain terminates withan aldehyde subgroup; a linear or branched, saturated or unsaturatedaliphatic chain having at least 2 carbons, and having at least oneketone subgroup; a linear or branched, saturated or unsaturatedaliphatic chain having at least 2 carbons; wherein the aliphatic chainterminates with a carboxylic acid subgroup; a linear or branched,saturated or unsaturated aliphatic chain having at least 2 carbons, andhaving at least one amide subgroup; a linear or branched, saturated orunsaturated aliphatic chain having at least 2 carbons, and having atleast one acyl group; a linear or branched, saturated or unsaturatedaliphatic chain having at least 2 carbons, and having at least onenitrogen containing moiety; a linear or branched, saturated orunsaturated aliphatic chain having at least 2 carbons, and having atleast one amine subgroup; a linear or branched, saturated or unsaturatedaliphatic chain having at least 2 carbons, and having at least one ethersubgroup; a linear or branched, saturated or unsaturated aliphatic chainhaving at least 2 carbons, and having at least one halogen subgroup; alinear or branched, saturated or unsaturated aliphatic chain having atleast 2 carbons, and having at least one nitronium subgroup; wherein R5is selected from the group consisting of: OH; NO₂; OR′; wherein R′ isselected from the group consisting of: a linear or branched, saturatedor unsaturated aliphatic chain having at least one carbon; a linear orbranched, saturated or unsaturated aliphatic chain having at least 2carbons, and having at least one hydroxyl subgroup; a linear orbranched, saturated or unsaturated aliphatic chain having at least 2carbons, and having at least one thiol subgroup; a linear or branched,saturated or unsaturated aliphatic chain having at least 2 carbons,wherein the aliphatic chain terminates with an aldehyde subgroup; alinear or branched, saturated or unsaturated aliphatic chain having atleast 2 carbons, and having at least one ketone subgroup; a linear orbranched, saturated or unsaturated aliphatic chain having at least 2carbons; wherein the aliphatic chain terminates with a carboxylic acidsubgroup; a linear or branched, saturated or unsaturated aliphatic chainhaving at least 2 carbons, and having at least one amide subgroup; alinear or branched, saturated or unsaturated aliphatic chain having atleast 2 carbons, and having at least one acyl group; a linear orbranched, saturated or unsaturated aliphatic chain having at least 2carbons, and having at least one nitrogen containing moiety; a linear orbranched, saturated or unsaturated aliphatic chain having at least 2carbons, and having at least one amine subgroup; a linear or branched,saturated or unsaturated aliphatic chain having at least 2 carbons, andhaving at least one halogen subgroup; a linear or branched, saturated orunsaturated aliphatic chain having at least 2 carbons, and having atleast one nitronium subgroup; wherein R6 is selected from the groupconsisting of: Hydrogen; NO₂; Cl; F; Br; I; SR′; and NR′₂; wherein R′ isdefined as above in R5; wherein R7 is selected from the group consistingof: Hydrogen; a linear or branched, saturated or unsaturated aliphaticchain having at least 2 carbons; and wherein R8 is an aliphatic cyclicgroup larger than benzene; wherein the larger than benzene comprises anychemical group containing 7 or more non-hydrogen atoms.

In preferred embodiments, the compound is:

In preferred embodiments, the benzodiazepine compound is anhydrous. Insome embodiments, the benzodiazepine compound has an orthorhombiccrystal structure.

In certain embodiments, the present invention provides a method oftreating an autoimmune disorder or hyperproliferative disordercomprising administering to a subject an effective amount of acomposition comprising an unsolvated compound.

DESCRIPTION OF THE FIGURES

FIG. 1 shows structural data of anhydrous Bz-423.

FIG. 2 shows powder x-ray diffraction data for anhydrous Bz-423.

FIG. 3 shows Raman spectroscopy data for anhydrous Bz-423.

FIG. 4 shows structural data of Bz-423 ethanol solvate.

FIG. 5 shows powder x-ray diffraction data for Bz-423 ethanol solvate.

FIG. 6 shows Raman spectroscopy data for Bz-423 ethanol solvate.

FIG. 7 shows Raman spectroscopy data for ball milled Bz-423 succinicacid (2:1).

FIG. 8 shows Raman spectroscopy data for ball milled Bz-423 citric acid(2:1).

FIG. 9 shows the structural data of Bz-423 biphenyl derivate.

FIG. 10 shows solubility (e.g., absorbance) as a function of time forunsolvated Bz-423, anhydrous Bz-423, Bz-423 acetic acid, and Bz-423citric acid.

FIG. 11 shows a comparison of ATP hydrolysis between unsolvated Bz-423and solvated Bz-423.

FIG. 12 shows a comparison of ATP synthesis between unsolvated Bz-423and solvated Bz-423.

FIG. 13 shows a comparison of cell viability between unsolvated Bz-423and solvated Bz-423.

FIG. 14 shows a UV-vis spectrum of Bz-423 in simulated gastric fluid.

FIG. 15 shows a UV-vis spectrum of Bz-423 in simulated gastric fluidbefore and after addition of K₂CO₃.

FIG. 16 shows Raman spectroscopy data for Bz-423 ethanol solvate.

FIG. 17 shows Raman spectroscopy data for Bz-423 1-propanol solvate.

FIG. 18 shows Raman spectroscopy data for Bz-423 2-propanol solvate.

FIG. 19 shows Raman spectroscopy data for Bz-423 1-butanol solvate.

FIG. 20 shows Raman spectroscopy data for Bz-423 2-butanol solvate.

FIG. 21 shows Raman spectroscopy data for Bz-423 1-pentanol solvate.

FIG. 22 shows Raman spectroscopy data for Bz-423 1-octanol solvate.

FIG. 23 shows Raman spectroscopy data for Bz-423 propylene glycolsolvate.

FIG. 24 shows Raman spectroscopy data for Bz-423 acetone glass.

DEFINITIONS

To facilitate an understanding of the present invention, a number ofterms and phrases are defined below.

As used herein, the term “benzodiazepine” refers to a seven memberednon-aromatic heterocyclic ring fused to a phenyl ring wherein theseven-membered ring has two nitrogen atoms, as part of the heterocyclicring. In some aspects, the two nitrogen atoms are in the 1 and 4positions or the 1 and 5 positions, as shown in the general structuresbelow:

The term “larger than benzene” refers to any chemical group containing 7or more non-hydrogen atoms.

As used herein, the term “polymorph” refers to a crystalline phase of asubstance. Many substances feature polymorphism, which is the ability ofa substance to exist as two or more crystalline phases that havedifferent arrangements and/or conformations of the molecules in thecrystal lattice. As used herein, the term polymorph includes amorphousphases and solvents/hydrates (i.e., psuedopolymorphs).

As used herein, the term “polymer library” refers to a compositioncomprising a plurality of different polymers positioned in particularlocations so as to allow reactions to occur on the polymers at theparticular locations. For example, containers or solid surfaces (e.g.,plate, glass, metal, or ceramic surfaces, multi-well plates, dishes,vials, tubes, flasks, etc.) with a plurality of different polymerscontained in discrete locations of the surface are polymer libraries.For example, a multi-well plate that contains a first polymer in a firstwell and a second polymer in a second well, etc. provides a polymerlibrary.

As used herein, the term “tabletability” refers to the capacity of apowdered material to be transformed into a tablet of specified strengthunder the effect of compaction pressure (Joiris et al., Pharm. Res.,15:1122 (1998); herein incorporated by reference in its entirety).Tabletability describes the effectiveness of the applied pressure inincreasing the tensile strength of the tablet and demonstrates therelationship between the cause, the compaction pressure, and the effect,the strength of the compact.

As used herein, the term “compressibility” refers to the ability of amaterial to undergo a reduction in volume as a result of an appliedpressure (Joiris et al., Pharm. Res., 15:1122 (1998); hereinincorporated by reference in its entirety). Compressibility indicatesthe ease with which a power bed undergoes volume reduction undercompaction pressure and is often represented by a plot showing thereduction of tablet porosity with increasing compaction pressure.

As used herein, the term “compactibility” refers to the ability of amaterial to produce tablets with sufficient strength under the effect ofdensification (Joiris et al., Pharm. Res., 15:1122 (1998); hereinincorporated by reference in its entirety). Compactibility shows thetensile strength of tablets normalized by tablet porosity. In manycases, the tensile strength decreases exponentially with increasingporosity (Ryshkewitch, J. Am. Cer. Soc., 36:65 (1953); hereinincorporated by reference in its entirety).

As used herein, the term “substituted aliphatic” refers to an alkanepossessing less than 10 carbons where at least one of the aliphatichydrogen atoms has been replaced by a halogen, an amino, a hydroxy, anitro, a thio, a ketone, an aldehyde, an ester, an amide, a loweraliphatic, a substituted lower aliphatic, or a ring (aryl, substitutedaryl, cycloaliphatic, or substituted cycloaliphatic, etc.). Examples ofsuch include, but are not limited to, 1-chloroethyl and the like.

As used herein, the term “substituted aryl” refers to an aromatic ringor fused aromatic ring system consisting of no more than three fusedrings at least one of which is aromatic, and where at least one of thehydrogen atoms on a ring carbon has been replaced by a halogen, anamino, a hydroxy, a nitro, a thio, a ketone, an aldehyde, an ester, anamide, a lower aliphatic, a substituted lower aliphatic, or a ring(aryl, substituted aryl, cycloaliphatic, or substituted cycloaliphatic).Examples of such include, but are not limited to, hydroxyphenyl and thelike.

As used herein, the term “cycloaliphatic” refers to a cycloalkanepossessing less than 8 carbons or a fused ring system consisting of nomore than three fused cycloaliphatic rings. Examples of such include,but are not limited to, decalin and the like.

As used herein, the term “substituted cycloaliphatic” refers to acycloalkane possessing less than 10 carbons or a fused ring systemconsisting of no more than three fused rings, and where at least one ofthe aliphatic hydrogen atoms has been replaced by a halogen, a nitro, athio, an amino, a hydroxy, a ketone, an aldehyde, an ester, an amide, alower aliphatic, a substituted lower aliphatic, or a ring (aryl,substituted aryl, cycloaliphatic, or substituted cycloaliphatic).Examples of such include, but are not limited to, 1-chlorodecalyl,bicyclo-heptanes, octanes, and nonanes (e.g., nonrbornyl) and the like.

As used herein, the term “heterocyclic” refers to a cycloalkane and/oran aryl ring system, possessing less than 8 carbons, or a fused ringsystem consisting of no more than three fused rings, where at least oneof the ring carbon atoms is replaced by oxygen, nitrogen or sulfur.Examples of such include, but are not limited to, morpholino and thelike.

As used herein, the term “substituted heterocyclic” refers to acycloalkane and/or an aryl ring system, possessing less than 8 carbons,or a fused ring system consisting of no more than three fused rings,where at least one of the ring carbon atoms is replaced by oxygen,nitrogen or sulfur, and where at least one of the aliphatic hydrogenatoms has been replaced by a halogen, hydroxy, a thio, nitro, an amino,a ketone, an aldehyde, an ester, an amide, a lower aliphatic, asubstituted lower aliphatic, or a ring (aryl, substituted aryl,cycloaliphatic, or substituted cycloaliphatic). Examples of suchinclude, but are not limited to 2-chloropyranyl.

As used herein, the term “linker” refers to a chain containing up to andincluding eight contiguous atoms connecting two different structuralmoieties where such atoms are, for example, carbon, nitrogen, oxygen, orsulfur. Ethylene glycol is one non-limiting example.

As used herein, the term “lower-alkyl-substituted-amino” refers to anyalkyl unit containing up to and including eight carbon atoms where oneof the aliphatic hydrogen atoms is replaced by an amino group. Examplesof such include, but are not limited to, ethylamino and the like.

As used herein, the term “lower-alkyl-substituted-halogen” refers to anyalkyl chain containing up to and including eight carbon atoms where oneof the aliphatic hydrogen atoms is replaced by a halogen. Examples ofsuch include, but are not limited to, chlorethyl and the like.

As used herein, the term “acetylamino” shall mean any primary orsecondary amino that is acetylated. Examples of such include, but arenot limited to, acetamide and the like.

The term “derivative” of a compound, as used herein, refers to achemically modified compound wherein the chemical modification takesplace either at a functional group of the compound or on the aromaticring. Non-limiting examples of 1,4-benzodiazepine derivatives of thepresent invention may include N-acetyl, N-methyl, N-hydroxy groups atany of the available nitrogens in the compound. Additional derivativesmay include those having a trifluoromethyl group on the phenyl ring.

The term “epidermal hyperplasia,” as used herein, refers to an abnormalmultiplication or increase in the number of normal cells in normalarrangement in epidermal tissue. Epidermal hyperplasia is acharacteristic of numerous disorders, including but not limited to,psoriasis.

The term “keratinocyte” as used herein, refers to a skin cell of thekeratinized layer of the epidermis.

The term “fibroblast” as used herein, refers to mesodermally derivedresident cells of connective tissue that secrete fibrillar procollagen,fibronectin and collegenase.

The term “pigment disorder” as used herein, refers to disordersinvolving skin pigment (e.g., melanin). Examples of pigment disordersinclude, but are not limited to, all forms of albinism, melasma, pigmentloss after skin damage, and vitiligo.

The term “stent” or “drug-eluting stent,” as used herein, refers to anydevice which when placed into contact with a site in the wall of a lumento be treated, will also place fibrin at the lumen wall and retain it atthe lumen wall. This can include especially devices deliveredpercutaneously to treat coronary artery occlusions and to sealdissections or aneurysms of splenic, carotid, iliac and poplitealvessels. The stent can also have underlying polymeric or metallicstructural elements onto which the fibrin is applied or the stent can bea composite of fibrin intermixed with a polymer. For example, adeformable metal wire stent such as that disclosed in U.S. Pat. No.4,886,062, herein incorporated by reference, could be coated with fibrinas set forth above in one or more coats (i.e., polymerization of fibrinon the metal framework by application of a fibrinogen solution and asolution of a fibrinogen-coagulating protein) or provided with anattached fibrin preform such as an encircling film of fibrin. The stentand fibrin could then be placed onto the balloon at a distal end of aballoon catheter and delivered by conventional percutaneous means (e.g.as in an angioplasty procedure) to the site of the restriction orclosure to be treated where it would then be expanded into contact withthe body lumen by inflating the balloon. The catheter can then bewithdrawn, leaving the fibrin stent of the present invention in place atthe treatment site. The stent may therefore provide both a supportingstructure for the lumen at the site of treatment and also a structuresupporting the secure placement of fibrin at the lumen wall. Generally,a drug-eluting stent allows for an active release of a particular drugat the stent implementation site.

As used herein, the term “catheter” refers generally to a tube used forgaining access to a body cavity or blood vessel.

As used herein, the term “valve” or “vessel” refers to any lumen withina mammal. Examples include, but are not limited to, arteries, veins,capillaries, and biological lumen.

As used herein, the term “restenosis” refers to any valve which isnarrowed. Examples include, but are not limited to, the reclosure of aperipheral or coronary artery following trauma to that artery caused byefforts to open a stenosed portion of the artery, such as, for example,by balloon dilation, ablation, atherectomy or laser treatment of theartery.

As used herein, “angioplasty” or “balloon therapy” or “balloonangioplasty” or “percutaneous transluminal coronary angioplasty” refersto a method of treating blood vessel disorders that involves the use ofa balloon catheter to enlarge the blood vessel and thereby improve bloodflow.

As used herein, “cardiac catheterization” or “coronary angiogram” refersto a test used to diagnose coronary artery disease using acatheterization procedure. Such a procedure may involve, for example,the injection of a contrast dye into the coronary arteries via acatheter, permitting the visualization of a narrowed or blocked artery.

As used herein, the term “subject” refers to organisms to be treated bythe methods of the present invention. Such organisms preferably include,but are not limited to, mammals (e.g., murines, simians, equines,bovines, porcines, canines, felines, and the like), and most preferablyincludes humans. In the context of the invention, the term “subject”generally refers to an individual who will receive or who has receivedtreatment (e.g., administration of benzodiazepine compound(s), andoptionally one or more other agents) for a condition characterized bythe dysregulation of apoptotic processes.

The term “diagnosed,” as used herein, refers to the recognition of adisease by its signs and symptoms (e.g., resistance to conventionaltherapies), or genetic analysis, pathological analysis, histologicalanalysis, and the like.

As used herein, the terms “anticancer agent,” or “conventionalanticancer agent” refer to any chemotherapeutic compounds, radiationtherapies, or surgical interventions, used in the treatment of cancer.

As used herein the term, “in vitro” refers to an artificial environmentand to processes or reactions that occur within an artificialenvironment. In vitro environments include, but are not limited to, testtubes and cell cultures. The term “in vivo” refers to the naturalenvironment (e.g., an animal or a cell) and to processes or reactionthat occur within a natural environment.

As used herein, the term “host cell” refers to any eukaryotic orprokaryotic cell (e.g., mammalian cells, avian cells, amphibian cells,plant cells, fish cells, and insect cells), whether located in vitro orin vivo.

As used herein, the term “cell culture” refers to any in vitro cultureof cells. Included within this term are continuous cell lines (e.g.,with an immortal phenotype), primary cell cultures, finite cell lines(e.g., non-transformed cells), and any other cell population maintainedin vitro, including oocytes and embryos.

In preferred embodiments, the “target cells” of the compositions andmethods of the present invention include, refer to, but are not limitedto, lymphoid cells or cancer cells. Lymphoid cells include B cells, Tcells, and granulocytes. Granulocyctes include eosinophils andmacrophages. In some embodiments, target cells are continuously culturedcells or uncultered cells obtained from patient biopsies.

Cancer cells include tumor cells, neoplastic cells, malignant cells,metastatic cells, and hyperplastic cells. Neoplastic cells can be benignor malignant. Neoplastic cells are benign if they do not invade ormetastasize. A malignant cell is one that is able to invade and/ormetastasize. Hyperplasia is a pathologic accumulation of cells in atissue or organ, without significant alteration in structure orfunction.

In one specific embodiment, the target cells exhibit pathological growthor proliferation. As used herein, the term “pathologically proliferatingor growing cells” refers to a localized population of proliferatingcells in an animal that is not governed by the usual limitations ofnormal growth.

As used herein, the term “un-activated target cell” refers to a cellthat is either in the G_(o) phase or one in which a stimulus has notbeen applied.

As used herein, the term “activated target lymphoid cell” refers to alymphoid cell that has been primed with an appropriate stimulus to causea signal transduction cascade, or alternatively, a lymphoid cell that isnot in G_(o) phase. Activated lymphoid cells may proliferate, undergoactivation induced cell death, or produce one or more of cytotoxins,cytokines, and other related membrane-associated proteins characteristicof the cell type. They are also capable of recognizing and binding anytarget cell that displays a particular antigen on its surface, andsubsequently releasing its effector molecules.

As used herein, the term “activated cancer cell” refers to a cancer cellthat has been primed with an appropriate stimulus to cause a signaltransduction. An activated cancer cell may or may not be in the G_(O)phase.

An activating agent is a stimulus that upon interaction with a targetcell results in a signal transduction cascade. Examples of activatingstimuli include, but are not limited to, small molecules, radiantenergy, and molecules that bind to cell activation cell surfacereceptors. Responses induced by activation stimuli can be characterizedby changes in, among others, intracellular Ca²⁺, superoxide, or hydroxylradical levels; the activity of enzymes like kinases or phosphatases; orthe energy state of the cell. For cancer cells, activating agents alsoinclude transforming oncogenes.

In one aspect, the activating agent is any agent that binds to a cellsurface activation receptor. These can be selected from the groupconsisting of, but not limited to, a T cell receptor ligand, a B cellactivating factor, a TNF, a Fas ligand, a proliferation inducing ligand,a cytokine, a chemokine, a hormone, an amino acid, a steroid, a B cellreceptor ligand, gamma irradiation, UV irradiation, an agent orcondition that enhances cell stress, or an antibody that specificallyrecognizes and binds a cell surface activation receptor. Antibodiesinclude monoclonal or polyclonal or a mixture thereof.

Examples of a T cell ligand include, but are not limited to, a peptidethat binds to an MHC molecule, a peptide MHC complex, or an antibodythat recognizes components of the T cell receptor.

Examples of a B cell ligand include, but are not limited to, a moleculeor antibody that binds to or recognizes components of the B cellreceptor.

Examples of agents or conditions that enhance cell stress include heat,radiation, oxidative stress, or growth factor withdrawal and the like.Examples of growth factors include, but are not limited to serum, IL-2,platelet derived growth factor (“PDGF”), and the like.

As used herein, the term “effective amount” refers to the amount of acompound (e.g., benzodiazepine) sufficient to effect beneficial ordesired results. An effective amount can be administered in one or moreadministrations, applications or dosages and is not limited intended tobe limited to a particular formulation or administration route.

As used herein, the term “dysregulation of the process of cell death”refers to any aberration in the ability of (e.g., predisposition) a cellto undergo cell death via either necrosis or apoptosis. Dysregulation ofcell death is associated with or induced by a variety of conditions,including for example, autoimmune disorders (e.g., systemic lupuserythematosus, rheumatoid arthritis, graft-versus-host disease,myasthenia gravis, Sjögren's syndrome, etc.), chronic inflammatoryconditions (e.g., psoriasis, asthma and Crohn's disease),hyperproliferative disorders (e.g., tumors, B cell lymphomas, T celllymphomas, etc.), viral infections (e.g., herpes, papilloma, HIV), andother conditions such as osteoarthritis and atherosclerosis.

It should be noted that when the dysregulation is induced by orassociated with a viral infection, the viral infection may or may not bedetectable at the time dysregulation occurs or is observed. That is,viral-induced dysregulation can occur even after the disappearance ofsymptoms of viral infection.

A “hyperproliferative disorder,” as used herein refers to any conditionin which a localized population of proliferating cells in an animal isnot governed by the usual limitations of normal growth. Examples ofhyperproliferative disorders include tumors, neoplasms, lymphomas andthe like. A neoplasm is said to be benign if it does not undergo,invasion or metastasis and malignant if it does either of these. Ametastatic cell or tissue means that the cell can invade and destroyneighboring body structures. Hyperplasia is a form of cell proliferationinvolving an increase in cell number in a tissue or organ, withoutsignificant alteration in structure or function. Metaplasia is a form ofcontrolled cell growth in which one type of fully differentiated cellsubstitutes for another type of differentiated cell. Metaplasia canoccur in epithelial or connective tissue cells. A typical metaplasiainvolves a somewhat disorderly metaplastic epithelium.

The pathological growth of activated lymphoid cells often results in anautoimmune disorder or a chronic inflammatory condition. As used herein,the term “autoimmune disorder” refers to any condition in which anorganism produces antibodies or immune cells which recognize theorganism's own molecules, cells or tissues. Non-limiting examples ofautoimmune disorders include autoimmune hemolytic anemia, autoimmunehepatitis, Berger's disease or IgA nephropathy, Celiac Sprue, chronicfatigue syndrome, Crohn's disease, dermatomyositis, fibromyalgia, graftversus host disease, Grave's disease, Hashimoto's thyroiditis,idiopathic thrombocytopenia purpura, lichen planus, multiple sclerosis,myasthenia gravis, psoriasis, rheumatic fever, rheumatic arthritis,scleroderma, Sjorgren syndrome, systemic lupus erythematosus, type 1diabetes, ulcerative colitis, vitiligo, and the like.

As used herein, the term “chronic inflammatory condition” refers to acondition wherein the organism's immune cells are activated. Such acondition is characterized by a persistent inflammatory response withpathologic sequelae. This state is characterized by infiltration ofmononuclear cells, proliferation of fibroblasts and small blood vessels,increased connective tissue, and tissue destruction. Examples of chronicinflammatory diseases include, but are not limited to, Crohn's disease,psoriasis, chronic obstructive pulmonary disease, inflammatory boweldisease, multiple sclerosis, and asthma. Autoimmune diseases such asrheumatoid arthritis and systemic lupus erythematosus can also result ina chronic inflammatory state.

As used herein, the term “co-administration” refers to theadministration of at least two agent(s) (e.g., benzodiazepines) ortherapies to a subject. In some embodiments, the co-administration oftwo or more agents/therapies is concurrent. In other embodiments, afirst agent/therapy is administered prior to a second agent/therapy.Those of skill in the art understand that the formulations and/or routesof administration of the various agents/therapies used may vary. Theappropriate dosage for co-administration can be readily determined byone skilled in the art. In some embodiments, when agents/therapies areco-administered, the respective agents/therapies are administered atlower dosages than appropriate for their administration alone. Thus,co-administration is especially desirable in embodiments where theco-administration of the agents/therapies lowers the requisite dosage ofa known potentially harmful (e.g., toxic) agent(s).

As used herein, the term “toxic” refers to any detrimental or harmfuleffects on a cell or tissue as compared to the same cell or tissue priorto the administration of the toxicant.

As used herein, the term “pharmaceutical composition” refers to thecombination of an active agent with a carrier, inert or active, makingthe composition especially suitable for diagnostic or therapeutic use invivo, in vivo or ex vivo.

As used herein, the term “pharmaceutically acceptable carrier” refers toany of the standard pharmaceutical carriers, such as a phosphatebuffered saline solution, water, emulsions (e.g., such as an oil/wateror water/oil emulsions), and various types of wetting agents. Thecompositions also can include stabilizers and preservatives. Forexamples of carriers, stabilizers and adjuvants. (See e.g., Martin,Remington's Pharmaceutical Sciences, 15th Ed., Mack Publ. Co., Easton,Pa. [1975]).

As used herein, the term “pharmaceutically acceptable salt” refers toany pharmaceutically acceptable salt (e.g., acid or base) of a compoundof the present invention which, upon administration to a subject, iscapable of providing a compound of this invention or an activemetabolite or residue thereof. As is known to those of skill in the art,“salts” of the compounds of the present invention may be derived frominorganic or organic acids and bases. Examples of acids include, but arenot limited to, hydrochloric, hydrobromic, sulfuric, nitric, perchloric,fumaric, maleic, phosphoric, glycolic, lactic, salicylic, succinic,toluene-p-sulfonic, tartaric, acetic, citric, methanesulfonic,ethanesulfonic, formic, benzoic, malonic, naphthalene-2-sulfonic,benzenesulfonic acid, and the like. Other acids, such as oxalic, whilenot in themselves pharmaceutically acceptable, may be employed in thepreparation of salts useful as intermediates in obtaining the compoundsof the invention and their pharmaceutically acceptable acid additionsalts.

Examples of bases include, but are not limited to, alkali metals (e.g.,sodium) hydroxides, alkaline earth metals (e.g., magnesium), hydroxides,ammonia, and compounds of formula NW₄ ⁺, wherein W is C₁₋₄ alkyl, andthe like.

Examples of salts include, but are not limited to: acetate, adipate,alginate, aspartate, benzoate, benzenesulfonate, bisulfate, butyrate,citrate, camphorate, camphorsulfonate, cyclopentanepropionate,digluconate, dodecylsulfate, ethanesulfonate, fumarate, flucoheptanoate,glycerophosphate, hemisulfate, heptanoate, hexanoate, hydrochloride,hydrobromide, hydroiodide, 2-hydroxyethanesulfonate, lactate, maleate,methanesulfonate, 2-naphthalenesulfonate, nicotinate, oxalate, palmoate,pectinate, persulfate, phenylpropionate, picrate, pivalate, propionate,succinate, tartrate, thiocyanate, tosylate, undecanoate, and the like.Other examples of salts include anions of the compounds of the presentinvention compounded with a suitable cation such as Na⁺, NH₄ ⁺, and NW₄⁺ (wherein W is a C₁₋₄ alkyl group), and the like.

For therapeutic use, salts of the compounds of the present invention arecontemplated as being pharmaceutically acceptable. However, salts ofacids and bases that are non-pharmaceutically acceptable may also finduse, for example, in the preparation or purification of apharmaceutically acceptable compound.

As used herein, the terms “solid phase supports” or “solid supports,”are used in their broadest sense to refer to a number of supports thatare available and known to those of ordinary skill in the art. Solidphase supports include, but are not limited to, silica gels, resins,derivatized plastic films, glass beads, cotton, plastic beads, aluminagels, and the like. As used herein, “solid supports” also includesynthetic antigen-presenting matrices, cells, liposomes, and the like. Asuitable solid phase support may be selected on the basis of desired enduse and suitability for various protocols. For example, for peptidesynthesis, solid phase supports may refer to resins such as polystyrene(e.g., PAM-resin obtained from Bachem, Inc., Peninsula Laboratories,etc.), POLYHIPE) resin (obtained from Aminotech, Canada), polyamideresin (obtained from Peninsula Laboratories), polystyrene resin graftedwith polyethylene glycol (TENTAGEL, Rapp Polymere, Tubingen, Germany) orpolydimethylacrylamide resin (obtained from Milligen/Biosearch,California).

As used herein, the term “pathogen” refers a biological agent thatcauses a disease state (e.g., infection, cancer, etc.) in a host.“Pathogens” include, but are not limited to, viruses, bacteria, archaea,fungi, protozoans, mycoplasma, prions, and parasitic organisms.

The terms “bacteria” and “bacterium” refer to all prokaryotic organisms,including those within all of the phyla in the Kingdom Procaryotae. Itis intended that the term encompass all microorganisms considered to bebacteria including Mycoplasma, Chlamydia, Actinomyces, Streptomyces, andRickettsia. All forms of bacteria are included within this definitionincluding cocci, bacilli, spirochetes, spheroplasts, protoplasts, etc.Also included within this term are prokaryotic organisms which are gramnegative or gram positive. “Gram negative” and “gram positive” refer tostaining patterns with the Gram-staining process which is well known inthe art. (See e.g., Finegold and Martin, Diagnostic Microbiology, 6thEd., CV Mosby St. Louis, pp. 13-15 [1982]). “Gram positive bacteria” arebacteria which retain the primary dye used in the Gram stain, causingthe stained cells to appear dark blue to purple under the microscope.“Gram negative bacteria” do not retain the primary dye used in the Gramstain, but are stained by the counterstain. Thus, gram negative bacteriaappear red.

As used herein, the term “microorganism” refers to any species or typeof microorganism, including but not limited to, bacteria, archaea,fungi, protozoans, mycoplasma, and parasitic organisms. The presentinvention contemplates that a number of microorganisms encompassedtherein will also be pathogenic to a subject.

As used herein, the term “fungi” is used in reference to eukaryoticorganisms such as the molds and yeasts, including dimorphic fungi.

As used herein, the term “virus” refers to minute infectious agents,which with certain exceptions, are not observable by light microscopy,lack independent metabolism, and are able to replicate only within aliving host cell. The individual particles (i.e., virions) typicallyconsist of nucleic acid and a protein shell or coat; some virions alsohave a lipid containing membrane. The term “virus” encompasses all typesof viruses, including animal, plant, phage, and other viruses.

The term “sample” as used herein is used in its broadest sense. A samplesuspected of indicating a condition characterized by the dysregulationof apoptotic function may comprise a cell, tissue, or fluids,chromosomes isolated from a cell (e.g., a spread of metaphasechromosomes), genomic DNA (in solution or bound to a solid support suchas for Southern blot analysis), RNA (in solution or bound to a solidsupport such as for Northern blot analysis), cDNA (in solution or boundto a solid support) and the like. A sample suspected of containing aprotein may comprise a cell, a portion of a tissue, an extractcontaining one or more proteins and the like.

As used herein, the terms “purified” or “to purify” refer, to theremoval of undesired components from a sample. As used herein, the term“substantially purified” refers to molecules that are at least 60% free,preferably 75% free, and most preferably 90%, or more, free from othercomponents with which they usually associated.

As used herein, the term “antigen binding protein” refers to proteinswhich bind to a specific antigen. “Antigen binding proteins” include,but are not limited to, immunoglobulins, including polyclonal,monoclonal, chimeric, single chain, and humanized antibodies, Fabfragments, F(ab′)2 fragments, and Fab expression libraries. Variousprocedures known in the art are used for the production of polyclonalantibodies. For the production of antibody, various host animals can beimmunized by injection with the peptide corresponding to the desiredepitope including but not limited to rabbits, mice, rats, sheep, goats,etc. In a preferred embodiment, the peptide is conjugated to animmunogenic carrier (e.g., diphtheria toxoid, bovine serum albumin(BSA), or keyhole limpet hemocyanin [KLH]). Various adjuvants are usedto increase the immunological response, depending on the host species,including but not limited to Freund's (complete and incomplete), mineralgels such as aluminum hydroxide, surface active substances such aslysolecithin, pluronic polyols, polyanions, peptides, oil emulsions,keyhole limpet hemocyanins, dinitrophenol, and potentially useful humanadjuvants such as BCG (Bacille Calmette-Guerin) and Corynebacteriumparvum.

For preparation of monoclonal antibodies, any technique that providesfor the production of antibody molecules by continuous cell lines inculture may be used (See e.g., Harlow and Lane, Antibodies: A LaboratoryManual, Cold Spring Harbor Laboratory Press, Cold Spring Harbor, N.Y.).These include, but are not limited to, the hybridoma techniqueoriginally developed by Köhler and Milstein (Köhler and Milstein,Nature, 256:495-497 [1975]), as well as the trioma technique, the humanB-cell hybridoma technique (See e.g., Kozbor et al., Immunol. Today,4:72 [1983]), and the EBV-hybridoma technique to produce humanmonoclonal antibodies (Cole et al., in Monoclonal Antibodies and CancerTherapy, Alan R. Liss, Inc.,pp. 77-96 [1985]).

According to the invention, techniques described for the production ofsingle chain antibodies (U.S. Pat. No. 4,946,778; herein incorporated byreference) can be adapted to produce specific single chain antibodies asdesired. An additional embodiment of the invention utilizes thetechniques known in the art for the construction of Fab expressionlibraries (Huse et al., Science, 246:1275-1281 [1989]) to allow rapidand easy identification of monoclonal Fab fragments with the desiredspecificity.

Antibody fragments that contain the idiotype (antigen binding region) ofthe antibody molecule can be generated by known techniques. For example,such fragments include but are not limited to: the F(ab′)2 fragment thatcan be produced by pepsin digestion of an antibody molecule; the Fab′fragments that can be generated by reducing the disulfide bridges of anF(ab′)2 fragment, and the Fab fragments that can be generated bytreating an antibody molecule with papain and a reducing agent.

Genes encoding antigen binding proteins can be isolated by methods knownin the art. In the production of antibodies, screening for the desiredantibody can be accomplished by techniques known in the art (e.g.,radioimmunoassay, ELISA (enzyme-linked immunosorbant assay), “sandwich”immunoassays, immunoradiometric assays, gel diffusion precipitinreactions, immunodiffusion assays, in situ immunoassays (using colloidalgold, enzyme or radioisotope labels, for example), Western Blots,precipitation reactions, agglutination assays (e.g., gel agglutinationassays, hemagglutination assays, etc.), complement fixation assays,immunofluorescence assays, protein A assays, and immunoelectrophoresisassays, etc.) etc.

As used herein, the term “immunoglobulin” or “antibody” refer toproteins that bind a specific antigen. Immunoglobulins include, but arenot limited to, polyclonal, monoclonal, chimeric, and humanizedantibodies, Fab fragments, F(ab′)₂ fragments, and includesimmunoglobulins of the following classes: IgG, IgA, IgM, IgD, IbE, andsecreted immunoglobulins (sIg). Immunoglobulins generally comprise twoidentical heavy chains and two light chains. However, the terms“antibody” and “immunoglobulin” also encompass single chain antibodiesand two chain antibodies.

The term “epitope” as used herein refers to that portion of an antigenthat makes contact with a particular immunoglobulin. When a protein orfragment of a protein is used to immunize a host animal, numerousregions of the protein may induce the production of antibodies whichbind specifically to a given region or three-dimensional structure onthe protein; these regions or structures are referred to as “antigenicdeterminants”. An antigenic determinant may compete with the intactantigen (i.e., the “immunogen” used to elicit the immune response) forbinding to an antibody.

The terms “specific binding” or “specifically binding” when used inreference to the interaction of an antibody and a protein or peptidemeans that the interaction is dependent upon the presence of aparticular structure (i.e., the antigenic determinant or epitope) on theprotein; in other words the antibody is recognizing and binding to aspecific protein structure rather than to proteins in general. Forexample, if an antibody is specific for epitope “A,” the presence of aprotein containing epitope A (or free, unlabelled A) in a reactioncontaining labeled “A” and the antibody will reduce the amount oflabeled A bound to the antibody.

As used herein, the terms “non-specific binding” and “backgroundbinding” when used in reference to the interaction of an antibody and aprotein or peptide refer to an interaction that is not dependent on thepresence of a particular structure (i.e., the antibody is binding toproteins in general rather that a particular structure such as anepitope).

As used herein, the term “modulate” refers to the activity of a compound(e.g., benzodiazepine compound) to affect (e.g., to promote or retard)an aspect of cellular function, including, but not limited to, cellgrowth, proliferation, apoptosis, and the like.

As used herein, the term “competes for binding” is used in reference toa first molecule (e.g., a first benzodiazepine derivative) with anactivity that binds to the same substrate (e.g., the oligomycinsensitivity conferring protein in mitochondrial ATP synthase) as does asecond molecule (e.g., a second benzodiazepine derivative or othermolecule that binds to the oligomycin sensitivity conferring protein inmitochondrial ATP synthase, etc.). The efficiency (e.g., kinetics orthermodynamics) of binding by the first molecule may be the same as, orgreater than, or less than, the efficiency of the substrate binding tothe second molecule. For example, the equilibrium binding constant(K_(D)) for binding to the substrate may be different for the twomolecules.

As used herein, the term “instructions for administering said compoundto a subject,” and grammatical equivalents thereof, includesinstructions for using the compositions contained in a kit for thetreatment of conditions characterized by the dysregulation of apoptoticprocesses in a cell or tissue (e.g., providing dosing, route ofadministration, decision trees for treating physicians for correlatingpatient-specific characteristics with therapeutic courses of action).The term also specifically refers to instructions for using thecompositions contained in the kit to treat autoimmune disorders (e.g.,systemic lupus erythematosus, rheumatoid arthritis, graft-versus-hostdisease, myasthenia gravis, Sjögren's syndrome, etc.), chronicinflammatory conditions (e.g., psoriasis, asthma and Crohn's disease),hyperproliferative disorders (e.g., tumors, B cell lymphomas, T celllymphomas, etc.), viral infections (e.g., herpes virus, papilloma virus,HIV), and other conditions such as osteoarthritis and atherosclerosis,and the like.

The term “test compound” refers to any chemical entity, pharmaceutical,drug, and the like, that can be used to treat or prevent a disease,illness, sickness, or disorder of bodily function, or otherwise alterthe physiological or cellular status of a sample (e.g., the level ofdysregulation of apoptosis in a cell or tissue). Test compounds compriseboth known and potential therapeutic compounds. A test compound can bedetermined to be therapeutic by using the screening methods of thepresent invention. A “known therapeutic compound” refers to atherapeutic compound that has been shown (e.g., through animal trials orprior experience with administration to humans) to be effective in suchtreatment or prevention. In preferred embodiments, “test compounds” areagents that modulate apoptosis in cells.

As used herein, the term “third party” refers to any entity engaged inselling, warehousing, distributing, or offering for sale a test compoundcontemplated for administered with a compound for treating conditionscharacterized by the dysregulation of apoptotic processes.

GENERAL DESCRIPTION OF THE INVENTION

Pharmaceutical companies expend much of their resources in attempts tofind new blockbuster drugs (greater than $1 billion/year sales) (D. EricWalters, BC 5220, Techniques in Biomedical Research, “The Rational Basisof Drug Design”). In order to be successful, a new drug should satisfyseveral criteria: safe to use; effective for the intended use; stable(chemically and metabolically); good solubility profile; syntheticallyfeasible; and novel (i.e., patentable). An important aspect of drugdevelopment is the identification of leads. A lead is any chemicalcompound that shows the biological activity sought. A lead is not thesame as a drug however—as it should meet the criteria listed above priorto use as a drug. There are two broad tasks in drug discovery. The firstis lead-finding. Here the task is to find a chemical compound that has adesired bioactivity. The second is lead-optimization, modifying the leadstructure to build in the other desirable properties (safety,solubility, stability, etc.).

There are many ways to find lead compounds. In the beginning, plants andother natural products were the source of most medicinal substances. Asthe science of medicinal chemistry evolved, it was discovered that theplants and natural products contained specific compounds that areresponsible for the therapeutic effect. It became possible to isolatethe active components, so that dosage could be more accuratelyregulated.

Other medicines came about because of accidental observations anddiscoveries (e.g., penicillin). The discovery of penicillin led to alarge-scale screening effort, in which thousands of soil microorganismswere grown and tested to see whether they could produce other substancesthat kill bacteria. Antibiotics such as streptomycin, neomycin,gentamicin, erythromycin, and the tetracyclines resulted from theseefforts.

Chemical modification of known drugs can often lead to improved drugs.For example, naturally occurring penicillin G is broken down bybacterial beta-lactamases. Addition of two —OCH₃ groups producesmethicillin, which is resistant to lactamase. Another example ofchemical modification is found in the opiate analgesics. The parentcompound is morphine, which occurs in opium poppies. Morphine is apowerful analgesic, but it has serious side effects: respiratorydepression, constipation, and dependence liability. Thousands of analogs(related chemical structures) have been synthesized in an effort to findanalgesics with lower incidence of side effects. In some cases, smallchanges in chemical structure may have a big influence on the activity.For example, nalorphine is a partial agonist (shows some morphine-likeactivity, and at higher concentration, antagonizes morphine effects),and naloxone is an antagonist. Considerable simplification of themolecule is possible. For example, meperidine has only two rings insteadof four, but it maintains strong analgesic activity. It has better oralabsorption than morphine, and shows less GI side effects. Methadone isan analgesic in which the original piperidine ring (6-membered ringcontaining a nitrogen atom) is completely absent. It retains analgesicactivity, has good oral activity, and has a long half-life in the body.Dextromethorphan is constructed on a mirror image of the morphine ringsystem. It has no opiate analgesic effects or side effects, but is auseful anti-tussive agent.

Some drugs are discovered by observing side effects of existing drugs.For example, minoxidil was found to grow hair on bald men as a sideeffect in a study of its antihypertensive effects. Viagra's effect onpenile dysfunction was discovered in clinical trials for treatment ofangina; it had originally been designed as an antihypertensive drug.

In the modern era, most leads are discovered using various screeningprocesses. For example, over a couple of decades, the National CancerInstitute has put hundreds of thousands of different chemical compoundsthrough a battery of anti-cancer assays. Current screening assays oftenemploy miniaturization and automation with robots for high throughputscreening, allowing hundreds of thousands of compounds to be screened ina short period of time.

Structure-based molecular design is yet another method to identify leadmolecules for drug design. This method is based on the premise thatdesired drug candidates possess significant structural and chemicalcomplementarity with their target molecules. This design method cancreate molecules with specific properties that make them conducive forbinding to the target site. The molecular structures that are designedby the structure-based design process are meant to interact withbiochemical targets, for example, whose three-dimensional structures areknown.

Even with the extensive resources expended in drug discovery and design,there are no systematic methods for generating drugs with desiredproperties. Thus, the art is in need of additional systems and methodsto facilitate the discovery and optimization of therapeutic and otheruseful compounds.

As a class of drugs, benzodiazepine compounds have been widely studiedand reported to be effective medicaments for treating a number ofdisease. For example, U.S. Pat. Nos. 4,076,823, 4,110,337, 4,495,101,4,751,223 and 5,776,946, each incorporated herein by reference in itsentirety, report that certain benzodiazepine compounds are effective asanalgesic and anti-inflammatory agents. Similarly, U.S. Pat. No.5,324,726 and U.S. Pat. No. 5,597,915, each incorporated by reference inits entirety, report that certain benzodiazepine compounds areantagonists of cholecystokinin and gastrin and thus might be useful totreat certain gastrointestinal disorders.

Other benzodiazepine compounds have been studied as inhibitors of humanneutrophil elastase in the treating of human neutrophilelastase-mediated conditions such as myocardial ischemia, septic shocksyndrome, among others (See e.g., U.S. Pat. No. 5,861,380 incorporatedherein by reference in its entirety). U.S. Pat. No. 5,041,438,incorporated herein by reference in its entirety, reports that certainbenzodiazepine compounds are useful as anti-retroviral agents.

Despite the attention benzodiazepine compounds have drawn, it willbecome apparent from the description below, that the present inventionprovides novel uses for benzodiazepine compounds and related and othercompounds and methods of using the compounds, as well as knowncompounds, for treating a variety of diseases.

Benzodiazepine compounds are known to bind to benzodiazepine receptorsin the central nervous system (CNS) and thus have been used to treatvarious CNS disorders including anxiety and epilepsy. Peripheralbenzodiazepine receptors have also been identified, which receptors mayincidentally also be present in the CNS. The present inventiondemonstrates that benzodiazepines and related compounds havepro-apoptotic and cytotoxic properties. The route of action of thesecompounds is not through the previously identified benzodiazepinereceptors.

Thus, in some embodiments, the present invention provides a number ofcompounds. In other embodiments, the present invention provides methodsfor using such compounds to regulate biological processes. The presentinvention also provides drug-screening methods to identify and optimizecompounds. In preferred embodiments, the present invention providesunsolvated benzodiazepine structures and benzodiazepine relatedstructures with long term storage capability, and stability under highpressures (e.g., storage pressures necessary in generating drugtablets). These and other research and therapeutic utilities aredescribed below.

DETAILED DESCRIPTION OF THE INVENTION

Exemplary compositions and methods of the present invention aredescribed in more detail in the following sections: I. Modulators ofCell Death; II. Modulators of Cell Growth and Proliferation; III.Benzodiazepine and Benzodiazepine Related Crystal Forms; IV.Pharmaceutical compositions, formulations, and exemplary administrationroutes and dosing considerations; V. Drug screens; VI. TherapeuticApplications; and VII. ATPase Inhibitors And Methods For IdentifyingTherapeutic Inhibitors.

The present invention herein incorporates by reference U.S. ProvisionalPatent Nos. 60/131,761, 60/165,511, 60/191,855, 60/312,560, 60/313,689,60/396,670, 60/565,788, 60/607,599, 60/641,040, and U.S. patentapplication Ser. Nos. 11/324,419, 11/176,719, 11/110,228, 10/935,333,10/886,450, 10/795,535, 10/634,114, 10/427,211, 10/427,212, 10/217,878,09/767,283, 09/700,101, and related applications. All compounds and usesdescribed in the above mentioned cases are contemplated to be part ofthe present invention. Additionally, all other known uses ofbenzodiazepines may be used with the new formulations of the invention.Additional references include, but are not limited to, Otto, M. W., etal., (2005) J. Clin. Psychiatry 66 Suppl. 2:34-38; Yoshii, M., et al.,(2005) Nippon Yakurigaku Zasshi 125(1):33-36; Yasuda, K. (2004) NipponRinsho. 62 Suppl. 12:360-363; Decaudin, D. (2004) 15(8):737-745; Bonnot,O., et al. (2003) Encephale. 29(6):553-559; Sugiyama, T. (2003)Ryoikibetsu Shokogun Shirizu. 40:489-492; Lacapere, J. J., et al.,(2003) Steroids. 68(7-8):569-585; Galiegue, S., et al., (2003) Curr.Med. Chem. 10(16):1563-1572; Papadopoulo, V. (2003) Ann. Pharm. Fr.61(1):30-50; Goethals, I., et al., (2002) Eur. J. Nucl. Med. Mol.Imaging 30(2):325-328; Castedo, M., et al., (2002) J. Exp. Med.196(9):1121-1125; Buffett-Jerrott, S. E., et al., (2002) Curr. Pham.Des. 8(1):45-58; Beurdeley-Thomas, A., et al., (2000) J. Nuerooncol.46(1):45-56; Smyth, W. F., et al., (1998) Electrophoresis19(16-17):2870-2882; Yoshii, M., et al., (1998) Nihon Shinkei SeishinYakurigaku Zasshi. 18(2):49-54; Trimble, M. and Hindmarch, I. (2000)Benzodiazepines, published by Wrighton Biomedical Publishing; andSalamone, S. J. (2001) Benzodiazepines and GHB—Detection andPharmacology, published by Humana Press; each herein incorporated byreference in their entireties.

The practice of the present invention employs, unless otherwiseindicated, conventional techniques of organic chemistry, pharmacology,molecular biology (including recombinant techniques), cell biology,biochemistry, and immunology, which are within the skill of the art.Such techniques are explained fully in the literature, such as,“Molecular cloning: a laboratory manual” Second Edition (Sambrook etal., 1989); “Oligonucleotide synthesis” (M. J. Gait, ed., 1984); “Animalcell culture” (R. I. Freshney, ed., 1987); the series “Methods inenzymology” (Academic Press, Inc.); “Handbook of experimentalimmunology” (D. M. Weir & C. C. Blackwell, eds.); “Gene transfer vectorsfor mammalian cells” (J. M. Miller & M. P. Calos, eds., 1987); “Currentprotocols in molecular biology” (F. M. Ausubel et al., eds., 1987, andperiodic updates); “PCR: the polymerase chain reaction” (Mullis et al.,eds., 1994); and “Current protocols in immunology” (J. E. Coligan etal., eds., 1991), each of which is herein incorporated by reference inits entirety.

I. Modulators of Cell Death

In preferred embodiments, the compounds of the present inventionregulate apoptosis through the exposure of cells to compounds. Theeffect of compounds can be measured by detecting any number of cellularchanges. Cell death may be assayed as described herein and in the art.In preferred embodiments, cell lines are maintained under appropriatecell culturing conditions (e.g., gas (CO₂), temperature and media) foran appropriate period of time to attain exponential proliferationwithout density dependent constraints. Cell number and or viability aremeasured using standard techniques, such as trypan blueexclusion/hemo-cytometry, or MTT dye conversion assay. Alternatively,the cell may be analyzed for the expression of genes or gene productsassociated with aberrations in apoptosis or necrosis.

In preferred embodiments, exposing the compounds of the presentinvention to a cell induces apoptosis. In some embodiments, the presentinvention causes an initial increase in cellular ROS levels (e.g., O₂⁻). In further embodiments, exposure of the compounds of the presentinvention to a cell causes an increase in cellular O₂ ⁻ levels. In stillfurther embodiments, the increase in cellular O₂ ⁻ levels resulting fromthe compounds of the present invention is detectable with aredox-sensitive agent that reacts specifically with O₂ ⁻ (e.g.,dihyroethedium (DHE)).

In other embodiments, increased cellular O₂ ⁻ levels resulting fromcompounds of the present invention diminish after a period of time(e.g., 10 minutes). In other embodiments, increased cellular O₂ ⁻ levelsresulting from the compounds of the present invention diminish after aperiod of time and increase again at a later time (e.g., 10 hours). Infurther embodiments, increased cellular O₂ ⁻ levels resulting from thecompounds of the present invention diminish at 1 hour and increase againafter 4 hours. In preferred embodiments, an early increase in cellularO₂ ⁻ levels, followed by a diminishing in cellular O₂ ⁻ levels, followedby another increase in cellular O₂ ⁻ levels resulting from the compoundsof the present invention is due to different cellular processes (e.g.,bimodal cellular mechanisms).

In some embodiments, the compounds of the present invention cause acollapse of a cell's mitochondrial ΔΨ_(m). In preferred embodiments, acollapse of a cell's mitochondrial ΔΨ_(m) resulting from the presentinvention is detectable with a mitochondria-selective potentiometricprobe (e.g., DiOC₆). In further embodiments, a collapse of a cell'smitochondrial ΔΨ_(m) resulting from the present invention occurs afteran initial increase in cellular O₂ ⁻ levels.

In some embodiments, the compounds of the present invention enablecaspace activation. In other embodiments, the compounds of the presentinvention cause the release of cytochrome c from mitochondria. Infurther embodiments, the compounds of the present invention altercystolic cytochrome c levels. In still other embodiments, alteredcystolic cytochrome c levels resulting from the compounds of the presentinvention are detectable with immunoblotting cytosolic fractions. Inpreferred embodiments, diminished cystolic cytochrome c levels resultingfrom the compounds of the present invention are detectable after aperiod of time (e.g., 10 hours). In further preferred embodiments,diminished cystolic cytochrome c levels resulting from the compounds ofthe present invention are detectable after 5 hours.

In other embodiments, the compounds of the present invention cause theopening of the mitochondrial PT pore. In preferred embodiments, thecellular release of cytochrome c resulting from the compounds of thepresent invention are consistent with a collapse of mitochondrialΔΨ_(m). In still further preferred embodiments, the compounds of thepresent invention cause an increase in cellular O₂ ⁻ levels after amitochondrial ΔΨ_(m) collapse and a release of cytochrome c. In furtherpreferred embodiments, a rise in cellular O₂ ⁻ levels is caused by amitochondrial ΔΨ_(m) collapse and release of cytochrome c resulting fromthe compounds of the present invention.

In other embodiments, the compounds of the present invention causecellular caspase activation. In preferred embodiments, caspaseactivation resulting from the compounds of the present invention ismeasurable with a pan-caspase sensitive fluorescent substrate (e.g.,FAM-VAD-fmk). In still further embodiments, caspase activation resultingfrom the compounds of the present invention tracks with a collapse ofmitochondrial ΔΨ_(m). In other embodiments, the compounds of the presentinvention cause an appearance of hypodiploid DNA. In preferredembodiments, an appearance of hypodiploid DNA resulting from thecompounds of the present invention is slightly delayed with respect tocaspase activation.

In some embodiments, the molecular target for the compounds of thepresent invention is found within mitochondria. In further embodiments,the molecular target of the compounds of the present invention involvesthe mitochondrial ATPase. The primary sources of cellular ROS includeredox enzymes and the mitochondrial respiratory chain (hereinafter MRC).In preferred embodiments, cytochrome c oxidase (complex IV of the MRC)inhibitors (e.g., NaN₃) preclude a dependent increase in cellular ROSlevels for the compounds of the present invention. In other preferredembodiments, the ubiquinol-cytochrome c reductase component of MRCcomplex III inhibitors (e.g., FK506) preclude a dependent increase inROS levels for the compounds of the present invention.

In some embodiments, an increase in cellular ROS levels due to thecompounds of the present invention result from the binding of thecompounds of the present invention to a target within mitochondria. Inpreferred embodiments, the compounds of the present invention oxidizes2′,7′-dichlorodihydrofluorescin (hereinafter DCF) diacetate to DCF. DCFis a redox-active species capable of generating ROS. In furtherembodiments, the rate of DCF production resulting from the presentinvention increases after a lag period.

Antimycin A generates O₂ ⁻ by inhibiting ubiquinol-cytochrome creductase. In preferred embodiments, the compounds of the presentinvention increase the rate of ROS production in an equivalent manner toantimycin A. In further embodiments, the compounds of the presentinvention increase the rate of ROS production in an equivalent manner toantimycin A under aerobic conditions supporting state 3 respiration. Infurther embodiments, the compounds of the present invention do notdirectly target the MPT pore. In additional embodiments, the compoundsof the present invention do not generate substantial ROS in thesubcellular S15 fraction (e.g., cytosol; microsomes). In even furtherembodiments, the compounds of the present invention do not stimulate ROSif mitochondria are in state 4 respiration.

MRC complexes I-III are the primary sources of ROS within mitochondria.In preferred embodiments, the primary source of an increase in cellularROS levels resulting from the dependent invention emanates from thesecomplexes as a result of inhibiting the mitochondrial F₁F₀-ATPase.Indeed, in still further embodiments, the present invention inhibitsmitochondrial ATPase activity of bovine sub-mitochondrial particles(hereinafter SMPs). In particularly preferred embodiments, the compoundsof the present invention bind to the OSCP component of the mitochondrialF₁F₀-ATPase.

Oligomycin is a macrolide natural product that binds to themitochondrial F₁F₀-ATPase, induces a state 3 to 4 transition, and as aresult, generates ROS (e.g., O₂ ⁻). In preferred embodiments, thecompounds of the present invention bind the OSCP component of themitochondrial F₁F₀-ATPase. In preferred embodiments, the compounds ofthe present invention bind the junction between the OSCP and the F₁subunit of the mitochondrial F₁F₀-ATPase. In some embodiments, thecompounds of the present invention bind the F₁ subunit. In certainembodiments, screening assays of the present invention permit detectionof binding partners of the OSCP, F₁, or OSCP/F₁ junction. OSCP is anintrinsically fluorescent protein. In certain embodiments, titrating asolution of test compounds of the present invention into an E. Colisample overexpressed with OSCP results in quenching of the intrinsicOSCP fluorescence. In other embodiments, fluorescent or radioactive testcompounds can be used in direct binding assays. In other embodiments,competition binding experiments can be conducted. In this type of assay,test compounds are assessed for their ability to compete with Bz-423 forbinding to the OSCP. In some embodiments, the compounds of the presentinvention cause a reduced increase in cellular ROS levels and reducedapoptosis in cells through regulation of the OSCP gene (e.g., alteringexpression of the OSCP gene). In further embodiments, the presentinvention functions by altering the molecular motions of the ATPasemotor.

II. Modulators of Cellular Proliferation and Cell Growth

In some embodiments, the compounds and methods of the present inventioncause descreased cellular proliferation. In other embodiments, thecompounds and methods of the present invention cause decreased cellularproliferation and apoptosis. For example, cell culture cytotoxicityassays conducted during the development of the present inventiondemonstrated that the compounds and methods of the present inventionprevents cell growth after an extended period in culture (e.g., 3 days).

III. Benzodiazepine and Benzodiazepine Related Crystal Forms

The present invention relates to systems and methods for generating newformulations of benzodiazepine compounds and benzodiazepine relatedcompounds. The present invention also provides high throughput systemsand methods for generating and identifying new crystallinebenzodiazepine and benzodiazepine related compounds.

For example, the present invention provides libraries of polymers fromwhich crystals are nucleated by exposing solutions (e.g., supersaturatedsolutions), the melt or vapor of the compound to the polymers. Growth ofcrystals on a plurality of polymers provides new methods for obtainingdesired polymorphs of compounds and for generating previouslyunidentified polymorphs of compounds. For example, the systems andmethods of the present invention have been used to identify novelpolymorphs of benzodiazepine compounds. For example, the systems andmethods have also been used to generate efficient methods for producingorthorhombic benzodiazepine compounds from solution. The novelpolymorphs identified by the systems and methods of the presentinvention find use in identifying drugs with enhanced properties,compared to previously available polymorphs of the compound. Thus, thesystems and methods of the present invention provide means for findingdrug leads and/or optimizing existing drugs (see, e.g., U.S. patentapplication Ser. No. 10/269,190; herein incorporated by reference in itsentirety).

Many pharmaceutical solids exhibit polymorphism (e.g., the ability of asubstance to exist as two or more crystalline phases that have differentarrangements and/or conformations of the molecules in the crystallattice). Because of their structural differences, polymorphs havedifferent solid-state properties. Consequently, polymorphism can exertprofound effects on pharmaceutical processing, including, but notlimited to, milling, granulation, and tableting (Conte et al., IIFarmaco (Ed. Pr.) 30:194 (1974); Otsuka et al., Chem. Pharm. Bull.,45:894 (1997); Otsuka et al., J. Pharm. Sci., 84:614 (1995); Tuladhar etal., J. Pharm. Pharmacol., 35:269 (1982); and Wong and Mitchell, Int. J.Pharm., 88:261 (1992)).

Despite the fact that upon dissolution, two polymorphs will yieldidentical solutions, the crystalline form affects the rate ofdissolution, equilibrium solubility, shelf life and ultimatelybioavailabilty. This has implications for isolation, clinical trials,and mass production and is therefore an important aspect of creating aviable therapeutic. With a greater number of polymorphs to choosebetween for a solid dosage, it is more likely that an optimal mixture ofproperties can be achieved leading to more efficacious drugs.

In its most simple form, the process of crystallization can beconsidered to start from a supersaturated solution, produced byevaporation, cooling, or addition of a nonsolvent, by formation ofnuclei. These species must achieve a sufficient size in order to proceedon to bulk crystals and it is the arrangement of the molecules in thesenanometer-sized structures that leads to the macroscopic crystal. Thusthe formation of unstable polymorphs can be attributed to their successin forming viable nuclei, a kinetic effect. Additives designed byconsideration of functional groups and lattice parameters (derived fromdiffraction methods) can also interact with these nuclei to stabilize ordestabilize them, and this approach of using designed additives has metwith success in some cases (Weissbuch et al., Acta Crystallogr. Sect.B-Struct. Sci. 51:115 (1995); Chen et al., J. Cryst. Growth 144:297(1994); and Davey et al., J. Am. Chem. Soc., 119:1767 (1997)). However,this method is best suited for modifying the crystallization behavior ofknown polymorphs and is not readily adapted to the generation of newforms with unknown lattice parameters.

Even in crystallizations where no additives are used, it is recognizedthat spontaneous (homogenous) nucleation is not very common, and in mostcases impurities on vessel walls function as heteronuclei to inducecrystal formation. The reluctance of saturated solutions to undergohomogeneous nucleation can be explained by the energetic barrier tobuilding a species with a high surface area to volume ratio where manyof the molecules do not experience the full stabilization of the bulk. Aheteronucleus reduces this barrier by providing stabilization of agrowing face of the crystal.

The present invention provides systems and methods for utilizing acombinatorial library of functionalized polymers for crystal formation.Both the types of functional groups and the spacing of these groups arealtered to produce surfaces that facilitate polymorph generation. Byvarying these parameters (e.g., systematically) throughout the library,these polymers produce crystal forms without prior knowledge of thepolymorph's structure and allow the discovery of new forms of compounds(e.g., pharmaceutical compounds, etc.) with improved properties overpreviously available structures. Properties that differ among polymorphsinclude, but are not limited to: packing properties (e.g., molar volumeand density, refractive index, electrical conductivity, thermalconductivity, hygroscopicity); thermodynamic properties (e.g., meltingand sublimation temperatures, internal [e.g., structural] energy,enthalpy, heat capacity, entropy, free energy and chemical potential,thermodynamic activity, vapor pressure, solubility); spectroscopicproperties (e.g., electronic transitions such as ultraviolet to visibleabsorption spectra, vibrational transitions such as infrared absorptionand Raman spectra, rotational transitions such as far infrared andmicrowave absorption spectra, nuclear spin transitions such as nuclearmagnetic resonance spectra); kinetic properties (e.g., dissolution rate,rates of solid state reactions, and stability); surface properties(e.g., surface free energy, interfacial tensions, habit); and mechanicalproperties (e.g., hardness, tensile strength, compactibility, tableting,handling, flow, and blending) (See e.g., “Polymorphism in PharmaceuticalSolids,” ed. Harry G. Brittain, Marcel Dekker, Inc., New York (1999)).

In some embodiments of the present invention, a plurality of polymersare provided with (e.g., placed onto or into) a solid surface or vesselto facilitate high throughput crystal growth and analysis. In someembodiments, the solid surface or vessel is a multi-chamber plate (e.g.,a 96-well or 384-well plate). However, the present invention is notlimited by the solid surface or vessel employed. As used herein, theterms “solid support” or “support” refer to any material that provides asolid or semi-solid structure with which another material (e.g., apolymer) can be associated. Such materials include smooth supports(e.g., metal, glass, plastic, silicon, and ceramic surfaces) as well astextured and porous materials. Such materials also include, but are notlimited to, gels, rubbers, polymers, and other non-rigid materials.Solid supports need not be flat. Supports include any type of shapeincluding spherical shapes (e.g., beads). Materials associated with thesolid support may be associated with any portion of the solid support(e.g., may be attached, enclosed, or in contact with an interior portionof a porous solid support material).

The present invention is not limited by the nature of the polymer usedto promote crystal growth. In preferred embodiments, the plurality ofpolymers used in screening methods of the present invention comprise twoor more polymers (e.g., three or more, four or more, five or more, . . ., ten or more, . . . , twenty or more, . . . , fifty or more differentpolymers). The maximum number of polymers employed in the systems andmethods of the present invention is constrained only by the availabilityof polymer materials (i.e., any of the thousands of known polymers maybe employed, as well as new polymers that are identified in the future)and by physical and space limitations of the testing area. However, thesystems and methods of the present invention may be employed at verylarge scales. For example, in some embodiments, 384-well plates are usedwherein the bottom surface of each well contains a different polymermaterial. Dozens of such plates may be arranged on shelves and dozens ofshelves may be placed in racks. A single laboratory space can holdhundreds of racks. Thus, a single room can house tens of millions ofdifferent polymers, wherein a solution with a candidate compound isapplied to each of the polymers and crystals are grown and analyzed toidentify the properties of the crystals. Further miniaturization allowseven more reactions to be run simultaneously in a single run.

While the present invention is not limited by the nature of the polymer,commercially available polymers that find use with the present inventioninclude, but are not limited to, acrylonitrile/butadiene/styrene resin,alginic acid (sodium salt), butyl/isobutyl methacrylate copolymer,cellulose acetate, cellulose acetate butyrate, cellulose propionate,cellulose triacetate, ethyl cellulose, ethylene/acrylic acid copolymer,ethylene/ethyl acrylate copolymer, ethylene/propylene copolymer,ethylene/vinyl acetate (14, 18, 25, 28, 33% and 40% VA) copolymer,hydroxybutyl methyl cellulose, hydroxypropyl cellulose, hydroxypropylmethyl cellulose, methyl cellulose, methyl vinyl ether/maleic acidcopolymer, methyl vinyl ether/maleic anhydride copolymer, nylon 6, nylon6/6, nylon 6/9, nylon 6/10, nylon 6/12, nylon 6(3)T, nylon 11, nylon 12,phenoxy resin, polyacetal, polyacrylamide, polyacrylamide carboxylmodified (low), polyacrylamide carboxyl modified (high), poly(acrylicacid), polyamide resin, 1,2-polybutadiene, poly(1-butene)isotactic,poly(n-butyl methacrylate), polycarbonate resin, poly(diallylisophthalate), poly(diallyl phthalate), poly(2,6-dimethyl-p-phenyleneoxide), poly(4,4-dipropoxy-2,2-diphenyl propane fumarate), poly(ethylmethacrylate), polyethylene high density, polyethylene low density,polyethylene chlorinated (25, 36, 42, and 48% chlorine), polyethylenechlorosulfonated, poly(ethylene oxide), polyethylene oxidized,poly(ethylene terephthalate), poly(2-hydroxyethyl methacrylate),poly(isobutyl methacrylate), polyisoprene chlorinated, poly(methylmethacrylate), poly(4-methyl-1-pentene), poly(alpha-methylstyrene),poly(p-phenylene ether-sulphone), poly(phenylene sulfide), polypropyleneisotactic chlorinated, polypropylene isotactic, polystyrene, polysulfoneresin, poly(tetrafluoroethylene), poly(2,4,6-tribromostyrene),poly(vinyl acetate), poly(vinyl alcohol) 100% hydrolyzed, poly(vinylalcohol) 98% hydrolyzed, poly(vinyl buyral), poly(vinyl chloride),poly(vinyl chloride) 1.8% carboxylated, poly(vinyl formal),polyvinylpyrrolidone, poly(vinyl stearate), poly(vinylidene fluoride),styrene/acrylonitrile copolymer (75/25), styrene/acrylonitrile copolymer(70/30), styrene/allyl alcohol copolymer, styrene/butadiene ABA blockcopolymer, styrene/butyl methacrylate copolymer,styrene/ethylene-butylene ABA block copolymer, styrene/maleic anhydridecopolymer, vinyl alcohol/vinyl butyral copolymer, vinyl chloride/vinylacetate (10, 12, and 19% VA) copolymer, vinyl chloride/vinyl acetatecopolymer 1% carboxylated, vinyl chloride/vinyl acetate/hydroxypropylacrylate terpolymer, and vinyl chloride/vinyl acetate/vinyl alcoholterpolymer, as well as, functionalized polybutadienes,poly(ethylene-co-propylene-co-5-methylene-2-norbornene),poly(perfluoropropylene oxide)-co-poly(perfluoroformaldehyde),metaldehyde, pectic acid, polyethylenimine, poly(ethylene-co-carbonmonoxide), poly(3-hydroxybutyric acid) and copolymers with valeric acid,polylactide, polyaminoacids, polyacenaphthylene, poly(dimethylsiloxane),poly[(dibenzo-18-crown-6)-co-formaldehyde] and other polymers containingmetal chelating groups, poly[(phenyl isocyanate)-co-formaldehyde],poly(vinylsulfonic acid), poly(melamine-co-formaldehyde),polyphosphates, polyphosphazenes, tributyltin fluoride, polysaccharides,and other organic and inorganic polymers.

Polymers at each location in the library can comprise a mixture of twoor more different polymers in one or more different locations. Thecombination of polymers in different ratios dramatically expands thediversity of conditions available in the libraries.

Solutions containing the compound to be screened are applied to thepolymers and incubated under conditions that facilitate crystal growth.The present invention is not limited by the manner in which thecompounds are applied to the polymers. In some embodiments, a solutionis used to supply each region of a polymer library. Solution may bedelivered by pouring, transfer through tubing, injection, or any othermeans. Where thousands to millions of individual polymers are used, inpreferred embodiments, an automated delivery system is used. The presentinvention is not limited to the use of solutions. Melts of materials andvapors onto the polymers also find use in the system and methods of thepresent invention.

The solvent used to solubilize any particular compound may be varied. Insome embodiments, a variety of solvents are used for each compound,wherein each different solvent type is exposed to each type of polymerto increase the range of crystallization conditions used in the library.In such embodiments, multiple regions (e.g., zones) of each polymer arecreated in the library to allow each solvent type to be combined witheach polymer type. In addition to different solvent types, a variety ofdifferent ingredients (e.g., salts) may be placed in the solution, yetfurther expanding the array of choices for library analysis. Suchapplications find use in the generation and isolation of newpseudopolymorphs (solvates and salts) that may be used as drugs.

Crystals formed on each polymer are analyzed using any suitable method.In some embodiments, analysis is conducted directly on the polymersurface, without removing the crystals. In other embodiments, crystalsare removed and analyzed. Analysis includes, but is not limited to,crystal structure analysis, analysis of spectroscopic, packing, density,thermodynamic, properties, kinetic, surface, and mechanical properties.In some embodiments, analysis includes functional analysis such astesting bioavailability or biological activity after administration to atest organism (e.g., an animal or plant). For example, in someembodiments, rapid screening is conducted using the D8 Discover withGADDS X-ray diffraction system (Bruker AXS, Madison, Wis.) or similarsystems.

Polymorphs identified in the screening method are compared to existingpolymorphs. Where a new polymorph is identified, the polymorph ischaracterized to identify properties that differ from previously knownpolymorphs (e.g., to identify improved drugs). Known polymorphsgenerated using the systems and methods of the present invention alsoare compared to existing production methods to identify whether thepolymer-based method of the present invention provides advantages overexisting production methods (e.g., less expensive or easier to produce,greater purity, superior crystals, ability to produce from aqueoussolution, etc.).

Polymorphs identified by the present invention can be produced in largequantities. In some embodiments, crystals are collected and used to seedfurther solutions of the compound. However, in some cases the presenceof the polymer surface may be required to generate crystals. In suchembodiments, large or multiple surfaces or vessels are provided with thepolymer known to generate the crystal to allow large-scale production.

Polymorphs produced by the methods of the present invention may be usedin the generation of pharmaceutical formulations. The novel polymorphsidentified increase the available choices for designing drugs withdesired properties, both in biological activity and in handling. Forexample, in order for many drugs to take action, they must dissolve inthe gut and be absorbed in the blood stream. In many cases the rate atwhich the drug dissolves can limit its effectiveness. The polymorphs ofthe present invention, either alone, or in combination with other formsof the drug find use in optimizing effectiveness, generally, or forparticular patients or patient groups (e.g., age groups, genders,species, etc.). In some cases, the novel polymorphs provide advantagesin shelf-life or the ability of the compound to be included in tablets(See e.g., Sun and Grant, Pharm. Res., 18:274 (2001)).

Exemplary benzodiazepine compounds provided by the present inventioninclude crystal forms and formulations of Bz-423:

Bz-423 differs from benzodiazepines in clinical use by the presence of ahydrophobic substituent at C-3. This substitution renders binding to theperipheral benzodiazepine receptor (“PBR”) weak (K_(d) ca. 1 μM) andprevents binding to the central benzodiazepine receptor so that Bz-423is not a sedative. Additionally, compositions of the present inventioncomprising benzodiazepine compounds (e.g., Bz-423) have been shown tobind to the oligomycin sensitivity conferring protein (OSCP) portion ofthe mitochondrial F₀F₁ ATPase synthase complex, to the OSCP junction, orto the F1 subunit (see, e.g., U.S. Provisional Patent Nos. 60/131,761,60/165,511, 60/191,855, 60/312,560, 60/313,689, 60/396,670, 60/565,788,60/607,599, 60/641,040, and U.S. patent application Ser. Nos.11/324,419, 11/176,719, 11/110,228, 10/935,333, 10/886,450, 10/795,535,10/634,114, 10/427,211, 10/427,212, 10/217,878, 09/767,283, 09/700,101,and related applications; each herein incorporated by reference in theirentireties).

Exemplary crystal forms of Bz-423 and related compounds provided by thepresent invention include, but are not limited to, anhydrous Bz-423,Bz-423 ethanol solvate, Bz-423 succinic acid (2:1), Bz-423 citric acid(2:1), Bz-423 biphenyl derivate, BZ-423-acetic acid, BZ-423-CH₃ CN,BZ-423-methanol, BZ-423-ethyl acetate, BZ-423-toluene, BZ-423-oxalicacid, BZ-423-fumaric acid, BZ-423-octanol, BZ-423-heptanoic acid,BZ-423-diphenyl ether, Bz-423-1-propanol solvate, Bz-423 2-propanolsolvate, Bz-423 1-butanol solvate, Bz-423 2-butanol solvate, Bz-4231-pentanol solvate, Bz-423 propylene glycol, Bz-423 acetone glass, andBZ-423-trichlorobenzene.

Additional exemplary compounds of the present invention also include,but are not limited to, crystal forms and formulations of:

or its enantiomer, wherein, R₁ is aliphatic or aryl; R₂ is aliphatic,aryl, —NH₂, —NHC(═O)—R₅; or a moiety that participates in hydrogenbonding, wherein R₅ is aryl, heterocyclic, —R₆—NH—C(═O)—R₇ or—R₆—C(═O)—NH—R₇, wherein R₆ is an aliphatic linker of 1-6 carbons and R₇is aliphatic, aryl, or heterocyclic, each of R₃ and R₄ is independentlya hydroxy, alkoxy, halo, amino, lower-alkyl-substituted-amino,acetylamino, hydroxyamino, an aliphatic group having 1-8 carbons and1-20 hydrogens, aryl, or heterocyclic; or a pharmaceutically acceptablesalt, prodrug or derivative thereof.

In the above structures, R₁ is a hydrocarbyl group of 1-20 carbons and1-20 hydrogens. Preferably, R₁ has 1-15 carbons, and more preferably,has 1-12 carbons. Preferably, R₁ has 1-12 hydrogens, and morepreferably, 1-10 hydrogens. Thus R₁ can be an aliphatic group or an arylgroup.

The term “aliphatic” represents the groups commonly known as alkyl,alkenyl, alkynyl, alicyclic. The term “aryl” as used herein represents asingle aromatic ring such as a phenyl ring, or two or more aromaticrings that are connected to each other (e.g., bisphenyl) or fusedtogether (e.g., naphthalene or anthracene). The aryl group can beoptionally substituted with a lower aliphatic group (e.g., C₁-C₄ alkyl,alkenyl, alkynyl, or C₃-C₆ alicyclic). Additionally, the aliphatic andaryl groups can be further substituted by one or more functional groupssuch as —NH₂, —NHCOCH₃, —OH, lower alkoxy (C₁-C₄), halo (—F, —Cl, —Br,or —I). It is preferable that R₁ is primarily a nonpolar moiety.

In the above structures, R₂ can be aliphatic, aryl, —NH₂, —NHC(═O)—R₅,or a moiety that participates in hydrogen bonding, wherein R₅, is aryl,heterocyclic, R₆—NH—C(═O)—R₇ or —R₆—C(═O)—NH—R₇, wherein R₆ is analiphatic linker of 1-6 carbons and R₇ is an aliphatic, aryl, orheterocyclic. The terms “aliphatic” and “aryl” are as defined above.

The term “a moiety that participates in hydrogen bonding” as used hereinrepresents a group that can accept or donate a proton to form a hydrogenbond thereby.

Some specific non-limiting examples of moieties that participate inhydrogen bonding include a fluoro, oxygen-containing andnitrogen-containing groups that are well-known in the art. Some examplesof oxygen-containing groups that participate in hydrogen bondinginclude: hydroxy, lower alkoxy, lower carbonyl, lower carboxyl, lowerethers and phenolic groups. The qualifier “lower” as used herein refersto lower aliphatic groups (C₁-C₄) to which the respectiveoxygen-containing functional group is attached.

Thus, for example, the term “lower carbonyl” refers to inter alia,formaldehyde, acetaldehyde.

Some nonlimiting examples of nitrogen-containing groups that participatein hydrogen bond formation include amino and amido groups. Additionally,groups containing both an oxygen and a nitrogen atom can alsoparticipate in hydrogen bond formation. Examples of such groups includenitro, N-hydroxy and nitrous groups.

It is also possible that the hydrogen-bond acceptor in the presentinvention can be the Π electrons of an aromatic ring. However, thehydrogen bond participants of this invention do not include those groupscontaining metal atoms such as boron. Further the hydrogen bonds formedwithin the scope of practicing this invention do not include thoseformed between two hydrogens, known as “dihydrogen bonds.” (See, R. H.Crabtree, Science, 282:2000-2001 [1998], for further description of suchdihydrogen bonds).

The term “heterocyclic” represents, for example, a 3-6 membered aromaticor nonaromatic ring containing one or more heteroatoms. The heteroatomscan be the same or different from each other. Preferably, at least oneof the heteroatom's is nitrogen. Other heteroatoms that can be presenton the heterocyclic ring include oxygen and sulfur.

Aromatic and nonaromatic heterocyclic rings are well-known in the art.Some nonlimiting examples of aromatic heterocyclic rings includepyridine, pyrimidine, indole, purine, quinoline and isoquinoline.Nonlimiting examples of nonaromatic heterocyclic compounds includepiperidine, piperazine, morpholine, pyrrolidine and pyrazolidine.Examples of oxygen containing heterocyclic rings include, but notlimited to furan, oxirane, 2H-pyran, 4H-pyran, 2H-chromene, andbenzofuran. Examples of sulfur-containing heterocyclic rings include,but are not limited to, thiophene, benzothiophene, and parathiazine.

Examples of nitrogen containing rings include, but not limited to,pyrrole, pyrrolidine, pyrazole, pyrazolidine, imidazole, imidazoline,imidazolidine, pyridine, piperidine, pyrazine, piperazine, pyrimidine,indole, purine, benzimidazole, quinoline, isoquinoline, triazole, andtriazine.

Examples of heterocyclic rings containing two different heteroatomsinclude, but are not limited to, phenothiazine, morpholine,parathiazine, oxazine, oxazole, thiazine, and thiazole.

The heterocyclic ring is optionally further substituted with one or moregroups selected from aliphatic, nitro, acetyl (i.e., —C(═O)—CH₃), oraryl groups.

Each of R₃ and R₄ can be independently a hydroxy, alkoxy, halo, amino,or substituted amino (such as lower-alkyl-substituted-amino, oracetylamino or hydroxyamino), or an aliphatic group having 1-8 carbonsand 1-20 hydrogens. When each of R₃ and R₄ is an aliphatic group, it canbe further substituted with one or more functional groups such as ahydroxy, alkoxy, halo, amino or substituted amino groups as describedabove. The terms “aliphatic” is defined above. Alternatively, each of R₃and R₄ can be hydrogen.

It is well-known that many 1,4-benzodiazepines exist as optical isomersdue to the chirality introduced into the heterocyclic ring at tile C₃position. The optical isomers are sometimes described as L- or D-isomersin the literature. Alternatively, the isomers are also referred to as R-and S-enantiomorphs. For the sake of simplicity, these isomers arereferred to as enantiomorphs or enantiomers. The 1,4-benzodiazepinecompounds described herein include their enantiomeric forms as well asracemic mixtures. Thus, the usage “benzodiazepine or its enantiomers”herein refers to the benzodiazepine as described or depicted, includingall its enantiomorphs as well as their racemic mixture.

From the above description, it is apparent that many specific examplesare represented by the generic formulas presented above. Thus, in oneexample, R₁ is aliphatic, R₂ is aliphatic, whereas in another example,R₁ is aryl and R₂ is a moiety that participates in hydrogen bondformation. Alternatively, R₁ can be aliphatic, and R₂ can be an—NHC(═O)—R₅, or a moiety that participates in hydrogen bonding, whereinR₅ is aryl, heterocyclic, —R₆—NH—C(═O)—R₇ or —R₆—C(═O)—NH—R₇, wherein R₆is an aliphatic linker of 1-6 carbons and R₇ is an aliphatic, aryl, orheterocyclic. A wide variety of sub combinations arising from selectinga particular group at each substituent position are possible and allsuch combinations are within the scope of this invention.

Additional exemplary compounds of the present invention also include,but are not limited to, crystal forms and formulations of:

enantiomers and pharmaceutically acceptable salts thereof:

-   -   wherein R₁ is an aliphatic group having 1 to 20 carbon atoms and        1 to 20 hydrogen atoms or an aryl group having up to 20 carbon        atoms and up to 20 hydrogen atoms;    -   wherein each of R₂ and R₃ is independently selected from the        group consisting of hydrogen, hydroxy, C₁₋₄alkoxy, halo, amino,        C₁₋₄alkyl-substituted-amino, acetylamino, hydroxyamino, an        aliphatic group having 1-8 carbons and 1-20 hydrogens, an aryl        group having from 6 to 14 carbon atoms, and a heterocyclic group        having a 3-6 membered aromatic or nonaromatic ring containing        one or more heteroatoms selected from nitrogen, oxygen, and        sulfur;    -   wherein R₄ is aliphatic or aryl;    -   wherein R₅ is selected from the group consisting of an aryl        group having from 6 to 14 carbon atoms, a heterocyclic group        having a 3-6 membered aromatic or nonaromatic ring containing        one or more heteroatoms selected from nitrogen, oxygen, and        sulfur, —R₆—NH—C(═O)—R₇, and —R₆—C(═O)—NH—R₇;    -   wherein R₆ is an aliphatic linker group of 1-6 carbons; and,

wherein R₇ is selected from the group consisting of an aliphatic grouphaving 1-8 carbons and 1-20 hydrogens, an aryl group having from 6 to 14carbon atoms and a heterocyclic group having a 3-6 membered aromatic ornonaromatic ring containing one or more heteroatoms selected fromnitrogen, oxygen, and sulfur.

In some preferred embodiments, crystal forms and formulations of thefollowing exemplary compound are provided:

In some preferred embodiments, crystal forms and formulations of thefollowing exemplary compounds are provided:

or its enantiomer, wherein, R₁ is aliphatic or aryl; R₂ is aliphatic,aryl, —NH₂, —NHC(═O)—R₅; or a moiety that participates in hydrogenbonding, wherein R₅ is aryl, heterocyclic, —R₆—NH—C(═O)—R₇ or—R₆—C(═O)—NH—R₇, wherein R₆ is an aliphatic linker of 1-6 carbons and R₇is aliphatic, aryl, or heterocyclic, each of R₃ and R₄ is independentlya hydroxy, alkoxy, halo, amino, lower-alkyl-substituted-amino,acetylamino, hydroxyamino, an aliphatic group having 1-8 carbons and1-20 hydrogens, aryl, or heterocyclic; or a pharmaceutically acceptablesalt, prodrug or derivative thereof.

In some preferred embodiments, crystal forms and formulations of thefollowing exemplary compounds are provided:

wherein R₂ is

and dimethylphenyl (all isomers) and ditrifluoromethyl (all isomers).

In some preferred embodiments, crystal forms and formulations of thefollowing exemplary compounds are provided:

In some preferred embodiments, crystal forms and formulations of thefollowing exemplary compounds are provided:

or a stereoisomer, a pharmaceutically-acceptable salt, hydrate, orprodrug thereof, wherein: R₁ and R₅ are attached to any available carbonatom of phenyl rings A and B, respectively, and at each occurrence areindependently selected from alkyl, substituted alkyl, halogen, cyano,nitro, OR₈, NR₈R₉, C(═O)R₈, CO₂R₈, C(═O)NR₈R₉, NR₈C(═O)R₉, NR₈C(═O)OR₉,S(O)₀R₉, NR₈SO₂R₉, SO₂NR₈R₉, cycloalkyl, heterocycle, aryl, andheteroaryl, and/or two of R₁ and/or two of R₅ join together to form afused benzo ring; R₂, R₃ and R₄ are independently selected fromhydrogen, alkyl, and substituted alkyl, or one of R₂, R₃ and R₄is a bondto R, T or Y and the other of R₂, R₃ and R₄ is selected from hydrogen,alkyl, and substituted alkyl; Z and Y are independently selected fromC(═O), —CO₂—, —SO₂—, —CH₂—, —CH₂C(═O)—, and —C(═O)C(═O)—, or Z may beabsent; R and T are selected from —CH₂—, —C(═O)—, and—CH[(CH₂)_(p)(Q)]-, wherein Q is NR₁₀R₁₁, OR₁₀ or CN; R₆ is selectedfrom alkyl, alkenyl, substituted alkyl, substituted alkenyl, aryl,cycloalkyl, heterocyclo, and heteroaryl; provided that where R₂ ishydrogen, Z-R₆ together are not —SO₂-Me or

R₇ is selected from hydrogen, alkyl, substituted alkyl, alkenyl,substituted alkenyl, aminoalkyl, halogen, cyano, nitro, keto(=O),hydroxy, alkoxy, alkylthio, C(═O)H, acyl, CO₂H, alkoxycarbonyl,carbamyl, sulfonyl, sulfonamidyl, cycloalkyl, heterocycle, aryl, andheteroaryl; R₈ and R₉ are independently selected from hydrogen, alkyl,substituted alkyl, cycloalkyl, heterocycle, aryl, and heteroaryl, or R₈and R₉ taken together to form a heterocycle or heteroaryl, except R₉ isnot hydrogen when attached to a sulfonyl group as in SO₂R₉; R₁₀ and R₁₁are independently selected from hydrogen, alkyl, and substituted alkyl;m and n are independently selected from 0, 1, 2 and 3; o, p and q areindependently 0, 1 or 2; and rand tare 0 or 1.

In further exemplary compounds, Z-R₆ taken together are selected from:i. thiophenyl optionally substituted with R₁₄; ii. imidazolyl optionallysubstituted with R₁₄; iii. —CH(aryl)(CO₂C₁₋₆alkyl); iv. —CO₂-alkyl; v.—SO₂-alkyl optionally substituted with up to three of halogen and/orphenyl; vi. —SO₂-alkenyl optionally substituted with phenyl; and vii.

wherein R₁₅ is halogen, alkyl, nitro, cyano, hydroxy, alkoxy,NHC(═O)alkyl, and/or two R₁₅ groups are taken together to form a fusedbenzo ring or a five to six membered heteroaryl; R₁₆ is selected fromhydrogen, halogen, alkyl, nitro, cyano, hydroxy, alkoxy, NHC(═O)alkyl,and phenyloxy or benzyloxy in turn optionally substituted with 1 to 3 ofhalogen, cyano, and C₁₋₄alkoxy; R₁₇ is selected from alkyl, alkoxy,CO₂C₁₋₆alkyl, and SO₂phenyl; and u and v are independently 0, 1 or 2.

In some preferred embodiments, crystal forms and formulations of thefollowing exemplary compounds are provided:

or a stereoisomer, a pharmaceutically-acceptable salt, hydrate, orprodrug thereof, in which: R₁ and R₅ are attached to any availablecarbon atom of phenyl ring A and phenyl ring B, respectively, and ateach occurrence are independently selected from C₁₋₆alkyl, substitutedC₁₋₆alkyl, halogen, cyano, O(C₁₋₆alkyl), O(phenyl), O(benzyl), NH₂,NH(C₁₋₆alkyl), N(C₁₋₆alkyl)₂, C(═O)H, C(═O)(C₁₋₆alkyl), CO₂H,CO₂(C₁₋₆alkyl), C(═O)NH₂, C(═O)NH(C₁₋₆alkyl), C(═O)N(C₁₋₆alkyl)₂,NHC(═O)(C₁₋₆alkyl), S(O)₂(C₁₋₆alkyl), NHSO₂(C₁₋₆alkyl), SO₂NH₂,SO₂NH(C₁₋₆alkyl), SO₂N(C₁₋₆alkyl)₂, C₃₋₇cycloalkyl, phenyl, five or sixmember heteroaryl, or four to seven membered heterocyclo, and/or two ofR₁ and/or two of R₅ join together to form a fused benzo ring; R₂ and R₃are independently selected from hydrogen and C₁₋₄alkyl; Z is —CO₂—,—SO₂—, or is absent; R₆ is selected from optionally-substituted alkyl,alkenyl, aryl, and heteroaryl; m and n are independently selected from0, 1, and 2; and q is 0 or 1.

In some preferred embodiments, crystal forms and formulations of thefollowing exemplary compounds are provided:

or a stereoisomer, a pharmaceutically-acceptable salt, hydrate, orprodrug thereof, wherein: R₁ and R₅ are attached to any available carbonatom of phenyl ring A and phenyl ring B, respectively, and at eachoccurrence are independently selected from alkyl, substituted alkyl,halogen, cyano, nitro, hydroxy, alkoxy, alkylthio, alkylamino, C(═O)H,acyl, CO₂H, alkoxycarbonyl, carbamyl, sulfonyl, sulfonamidyl,cycloalkyl, heterocycle, aryl, and heteroaryl, and/or two of R₁ and/ortwo of R₅ join together to form a fused benzo ring; R₂, R₃ and R₄ areindependently selected from hydrogen and alkyl; Z is —CO₂—, —SO₂—, or isabsent; R₆ is selected from: a) C₁₋₄alkyl or C₁₋₄alkenyl optionallysubstituted with up to three of halogen, aryl and CO₂C₁₋₆alkyl; b)phenyl optionally substituted with up to three R₁₂ and/or having fusedthereto a benzo-ring or a five to six membered heteroaryl; c) heteroarylselected from thiophenyl, imidazolyl, pyrazolyl, and isoxazolyl, whereinsaid heteroaryl is optionally substituted with up to two R₁₂, providedthat where R₂ ishydrogen, Z-R₆ together are not —SO₂—Me or

R₇ is selected from hydrogen, keto(═O), C₁₋₆alkyl, substitutedC₁₋₆alkyl, halogen, cyano, O(C₁₋₆alkyl), O(phenyl), O(benzyl), NH₂,NH(C₁₋₆alkyl), N(C₁₋₆alkyl)₂, C(═O)H, C(═O)(C₁₋₆alkyl), CO₂H,CO₂(C₁₋₆alkyl-); R₁₂ at each occurrence is independently selected fromeach other R₁₂ from the group consisting of C₁₋₆alkyl, halogen, nitro,cyano, hydroxy, alkoxy, NHC(═O)alkyl, —CO₂alkyl, —SO₂phenyl, five to sixmembered monocyclic heteroaryl, and phenyloxy or benzyloxy in turnoptionally substituted with halogen, C₁₋₄alkyl, and/or O(C₁₋₄alkyl); andm and n are independently selected from 0, 1, or 2.

In further exemplary compounds, Z is —SO₂—; R₆ is selected fromC₁₋₄alkyl, trifluoromethyl, benzyl, C₂₋₃alkenyl substituted with phenyl,

R₁₅ is halogen, alkyl, nitro, cyano, hydroxy, alkoxy, NHC(═O)alkyl,and/or two R₁₅ groups are taken together to form a fused benzo ring or afive to six membered heteroaryl; R₁₆ is selected from hydrogen, halogen,alkyl, nitro, cyano, hydroxy, alkoxy, NHC(═O)alkyl, and phenyloxy orbenzyloxy in turn optionally substituted with 1 to 3 of halogen, cyano,and C₁₋₄alkoxy; R₁₇ is selected from alkyl, alkoxy, CO₂C₁₋₆alkyl, andSO₂phenyl; and u and v are independently 0, 1 or 2.

In some preferred embodiments, crystal forms and formulations of thefollowing exemplary compounds are provided:

or a stereoisomer, a pharmaceutically-acceptable salt, hydrate, orprodrug thereof, wherein: R₁ is selected from the group consisting of H,CN and SO₂-piperidine; R₂ is selected from the group consisting of H,4-Cl-Ph, Ph, and 2-Me-imidazole; R₃ is selected from the groupconsisting of H, CH₂-2-imidazole, and CH2-2-oxazole.

In some preferred embodiments, crystal forms and formulations of thefollowing exemplary compounds are provided:

or a stereoisomer, a pharmaceutically-acceptable salt, hydrate, orprodrug thereof, wherein: R₁ is selected from the group consisting of H,2,4-Cl₂, 2-4-Me₂, and 2,5-(CF₃)₂; R₂ is selected from the groupconsisting of H, 4-Cl, 4-Me, 2,4-Cl₂, 2,4-Me₂, 3-Cl; X is selected fromthe group consisting of O and NH; Y is selected from the groupconsisting of S, O, NCN, CO(3-CN-Ph), CO(4-CN-Ph), CO(4-Cl-Ph), andCOEt.

In some preferred embodiments, crystal forms and formulations of thefollowing exemplary compounds are provided:

Additional exemplary compounds of the present invention are described inU.S. Provisional Patent Nos. U.S. Provisional Patent Nos. 60/131,761,60/165,511, 60/191,855, 60/312,560, 60/313,689, 60/396,670, 60/565,788,60/607,599, 60/641,040, and U.S. patent application Ser. Nos.11/324,419, 11/176,719, 11/110,228, 10/935,333, 10/886,450, 10/795,535,10/634,114, 10/427,211, 10/427,212, 10/217,878, 09/767,283, 09/700,101,and related applications; each herein incorporated by reference in theirentireties.

In some preferred embodiments, crystal forms and formulations of thefollowing exemplary compounds are provided:

R1 is H or hydroxyEach of R2 through R6 may be the same or different and is selected fromhydrogen, a hydroxy, an alkoxy, a halo, an amino, a lower-alkyl-asubstituted-amino, an acetylamino, a hydroxyamino, an aliphatic grouphaving 1-8 carbons and 1-20 hydrogens, a substituted aliphatic group ofsimilar size, a cycloaliphatic group consisting of <10 carbons, asubstituted cycloaliphatic group, an aryl, and a heterocyclic

Each of R1 through R10 maybe the same or different and is selected fromhydrogen, a hydroxy, an alkoxy, a halo, an amino, a lower-alkyl-asubstituted-amino, an acetylamino, a hydroxyamino, an aliphatic grouphaving 1-8 carbons and 1-20 hydrogens, a substituted aliphatic group ofsimilar size, a cycloaliphatic group consisting of <10 carbons, asubstituted cycloaliphatic group, an aryl, and a heterocyclic

Each of R1 through R11 may be the same or different and is selected fromhydrogen, a hydroxy, an alkoxy, a halo, an amino, a lower-alkyl-asubstituted-amino, an acetylamino, a hydroxyamino, an aliphatic grouphaving 1-8 carbons and 1-20 hydrogens, a substituted aliphatic group ofsimilar size, a cycloaliphatic group consisting of <10 carbons, asubstituted cycloaliphatic group, an aryl, and a heterocyclic

Each of R1 through R10 may be the same or different and is selected fromhydrogen, a hydroxy, an alkoxy, a halo, an amino, a lower-alkyl-asubstituted-amino, an acetylamino, a hydroxyamino, an aliphatic grouphaving 1-8 carbons and 1-20 hydrogens, a substituted aliphatic group ofsimilar size, a cycloaliphatic group consisting of <10 carbons, asubstituted cycloaliphatic group, an aryl, and a heterocyclic

Each of R1 through R10 may be the same or different and is selected fromhydrogen, a hydroxy, an alkoxy, a halo, an amino, a lower-alkyl-asubstituted-amino, an acetylamino, a hydroxyamino, an aliphatic grouphaving 1-8 carbons and 1-20 hydrogens, a substituted aliphatic group ofsimilar size, a cycloaliphatic group consisting of <10 carbons, asubstituted cycloaliphatic group, an aryl, and a heterocyclic

Each of R1 through R6 may be the same or different and is selected fromhydrogen, a hydroxy, an alkoxy, a halo, an amino, a lower-alkyl-asubstituted-amino, an acetylamino, a hydroxyamino, an aliphatic grouphaving 1-8 carbons and 1-20 hydrogens, a substituted aliphatic group ofsimilar size, a cycloaliphatic group consisting of <10 carbons, asubstituted cycloaliphatic group, an aryl, and a heterocyclic

wherein R₁ is selected from napthalalanine; phenol; 1-Napthalenol;2-Napthalenol;

and quinolines.

In some preferred embodiments, compositions comprising crystal forms andformulations of the following exemplary compounds are provided:

wherein R₁ is selected from:

The stereochemistry of all derivatives embodied in the present inventionis R, S, or racemic.

In some preferred embodiments, compositions comprising crystal forms andformulations of the following exemplary compound are provided:

In some preferred embodiments, compositions comprising crystal forms andformulations of the following exemplary compounds are provided:

wherein R1, R2, R3 and R4 are selected from the group consisting of:hydrogen; CH₃; a linear or branched, saturated or unsaturated aliphaticchain having at least 2 carbons; a linear or branched, saturated orunsaturated aliphatic chain having at least 2 carbons, and having atleast one hydroxy subgroup; a linear or branched, saturated orunsaturated aliphatic chain having at least 2 carbons, and having atleast one thiol subgroup; a linear or branched, saturated or unsaturatedaliphatic chain having at least 2 carbons, wherein said aliphatic chainterminates with an aldehyde subgroup; a linear or branched, saturated orunsaturated aliphatic chain having at least 2 carbons, and having atleast one ketone subgroup; a linear or branched, saturated orunsaturated aliphatic chain having at least 2 carbons; wherein saidaliphatic chain terminates with a carboxylic acid subgroup; a linear orbranched, saturated or unsaturated aliphatic chain having at least 2carbons, and having at least one amide subgroup; a linear or branched,saturated or unsaturated aliphatic chain having at least 2 carbons, andhaving at least one acyl group; a linear or branched, saturated orunsaturated aliphatic chain having at least 2 carbons, and having atleast one nitrogen containing moiety (e.g.,nitro, nitrile, etc.); alinear or branched, saturated or unsaturated aliphatic chain having atleast 2 carbons, and having at least one amine subgroup; a linear orbranched, saturated or unsaturated aliphatic chain having at least 2carbons, and having at least one ether subgroup; a linear or branched,saturated or unsaturated aliphatic chain having at least 2 carbons, andhaving at least one halogen subgroup; a linear or branched, saturated orunsaturated aliphatic chain having at least 2 carbons, and having atleast one nitronium subgroup; wherein R5 is selected from the groupconsisting of: OH; NO2; NR′; OR′; wherein R′ is selected from the groupconsisting of: a linear or branched, saturated or unsaturated aliphaticchain having at least one carbon; a linear or branched, saturated orunsaturated aliphatic chain having at least 2 carbons, and having atleast one hydroxyl subgroup; a linear or branched, saturated orunsaturated aliphatic chain having at least 2 carbons, and having atleast one thiol subgroup; a linear or branched, saturated or unsaturatedaliphatic chain having at least 2 carbons, wherein said aliphatic chainterminates with an aldehyde subgroup; a linear or branched, saturated orunsaturated aliphatic chain having at least 2 carbons, and having atleast one ketone subgroup; a linear or branched, saturated orunsaturated aliphatic chain having at least 2 carbons; wherein saidaliphatic chain terminates with a carboxylic acid subgroup; a linear orbranched, saturated or unsaturated aliphatic chain having at least 2carbons, and having at least one amide subgroup; a linear or branched,saturated or unsaturated aliphatic chain having at least 2 carbons, andhaving at least one acyl group; a linear or branched, saturated orunsaturated aliphatic chain having at least 2 carbons, and having atleast one nitrogen containing moiety (e.g., nitro, nitrile, etc.); alinear or branched, saturated or unsaturated aliphatic chain having atleast 2 carbons, and having at least one amine subgroup; a linear orbranched, saturated or unsaturated aliphatic chain having at least 2carbons, and having at least one halogen subgroup; a linear or branched,saturated or unsaturated aliphatic chain having at least 2 carbons, andhaving at least one nitronium subgroup; wherein R6 is selected from thegroup consisting of: Hydrogen; NO₂; Cl; F; Br; I; SR′; and NR′₂; whereinR′ is defined as above in R5; wherein R7 is selected from the groupconsisting of: Hydrogen; a linear or branched, saturated or unsaturatedaliphatic chain having at least 2 carbons; and wherein R8 is analiphatic cyclic group larger than benzene; wherein said larger thanbenzene comprises any chemical group containing 7 or more non-hydrogenatoms, and is an aryl or aliphatic cyclic group. In some embodiments, R′is any functional group that protects the oxygen of R5 from metabolismin vivo, until the compound reaches its biological target (e.g.,mitochondria). In some embodiments, R′ protecting group(s) ismetabolized at the target site, converting R5 to a hydroxyl group.

In some preferred embodiments, crystal forms and formulations of thefollowing exemplary compounds are provided:

including both R and S enantiomeric forms and racemic mixtures;wherein R1 comprises a chemical moiety comprising a hydrogen bondingproton donor (e.g., a hydroxyl group, a phenol group, an amide group, asulfonamide group, an amine group, an aniline group, a benzimidizalonegroup, a carbamate group, and an imidizole group); and R2 comprises ahydrophobic chemical moiety.

In preferred embodiments, R1 is selected from the group consisting of:

wherein R1′, R2, R3 and R4 are selected from the group consisting of:hydrogen; CH₃; a linear or branched, saturated or unsaturated aliphaticchain having at least 1 carbon; a linear or branched, saturated orunsaturated aliphatic chain having at least 2 carbons and at least onehydroxy subgroup; a linear or branched, saturated or unsaturated,substituted or non-substituted, aliphatic chain having at least 2carbons and having at least one thiol subgroup; a linear or branched,saturated or unsaturated, substituted or non-substituted, aliphaticchain having at least 2 carbons wherein the aliphatic chain terminateswith an aldehyde subgroup; a linear or branched, saturated orunsaturated aliphatic chain having at least 2 carbons, and having atleast one ketone subgroup; a linear or branched, saturated orunsaturated, substituted or non-substituted, aliphatic chain having atleast 2 carbons; wherein the aliphatic chain terminates with acarboxylic acid subgroup; a linear or branched, saturated orunsaturated, substituted or non-substituted, aliphatic chain having atleast 2 carbons, and having at least one amide subgroup; a linear orbranched, saturated or unsaturated, substituted or non-substituted,aliphatic chain having at least 2 carbons, and having at least one acylgroup; a linear or branched, saturated or unsaturated, substituted ornon-substituted, aliphatic chain having at least 2 carbons, and havingat least one nitrogen containing moiety; a linear or branched, saturatedor unsaturated, substituted or non-substituted, aliphatic chain havingat least 2 carbons, and having at least one amine subgroup; a linear orbranched, saturated or unsaturated, substituted or non-substituted,aliphatic chain having at least 2 carbons, and having at least one ethersubgroup; a linear or branched, saturated or unsaturated, substituted ornon-substituted, aliphatic chain having at least 2 carbons, and havingat least one halogen subgroup; a linear or branched, saturated orunsaturated, substituted or non-substituted, aliphatic chain having atleast 2 carbons, and having at least one nitronium subgroup; and R5 isOH.

In preferred embodiments, R2 is selected from group consisting of:napthalalanine; phenol; 1-Napthalenol; 2-Napthalenol;

In some preferred embodiments, R1 is selected from the group consistingof:

In some preferred embodiments, crystal forms and formulations of thefollowing exemplary compounds are provided:

wherein R3 is selected from the group consisting of Hydrogen; amino; anda linear or branched, saturated or unsaturated, substituted (e.g.,substituted with amines, esters, amides or phosphatases) ornon-substituted, aliphatic chain having at least 2 carbons;

R4 is selected from the group consisting of H, a ketone, and a nitrogen;and

R5 is selected from H, a hydroxy, an alkoxy, a carboxylic acid, acarboxylic ester, a halogen, a nitro, a sulfonamide, an amide, acarbamate, an amino, a lower-alkyl, a substituted-amino, an acetylamino,a hydroxyamino, an aliphatic group having 1-8 carbons and 1-20hydrogens, a substituted aliphatic group of similar size, acycloaliphatic group consisting of less than 10 carbons, a substitutedcycloaliphatic group, an aryl, a heterocyclic, NO₂; SR′; and NR′₂,wherein R′ is defined as a linear or branched, saturated or unsaturatedaliphatic chain having at least one carbon; a linear or branched,saturated or unsaturated aliphatic chain having at least 2 carbons, andhaving at least one hydroxyl subgroup; a linear or branched, saturatedor unsaturated aliphatic chain having at least 2 carbons, and having atleast one thiol subgroup; a linear or branched, saturated or unsaturatedaliphatic chain having at least 2 carbons, wherein the aliphatic chainterminates with an aldehyde subgroup; a linear or branched, saturated orunsaturated aliphatic chain having at least 2 carbons, and having atleast one ketone subgroup; a linear or branched, saturated orunsaturated aliphatic chain having at least 2 carbons; wherein thealiphatic chain terminates with a carboxylic acid subgroup; a linear orbranched, saturated or unsaturated aliphatic chain having at least 2carbons, and having at least one amide subgroup; a linear or branched,saturated or unsaturated aliphatic chain having at least 2 carbons, andhaving at least one acyl group; a linear or branched, saturated orunsaturated aliphatic chain having at least 2 carbons, and having atleast one nitrogen containing moiety; a linear or branched, saturated orunsaturated aliphatic chain having at least 2 carbons, and having atleast one amine subgroup; a linear or branched, saturated or unsaturatedaliphatic chain having at least 2 carbons, and having at least onehalogen subgroup; a linear or branched, saturated or unsaturatedaliphatic chain having at least 2 carbons, and having at least onenitronium subgroup.

In other preferred embodiments R2 is any chemical group that permits thecompound to bind to OSCP. In some such embodiments, R2 comprises ahydrophobic aromatic group. In preferred embodiments R2 comprises ahydrophobic aromatic group larger than benzene (e.g., a benzene ringwith non-hydrogen substituents, a moiety having two or more aromaticrings, a moiety with 7 or more carbon atoms, etc.).

In some preferred embodiments, crystal forms and formulations of thefollowing exemplary compounds are provided:

wherein R₂ is

and dimethylphenyl (all isomers) and ditrifluoromethyl (all isomers).

In some preferred embodiments, crystal forms and formulations of thefollowing exemplary compounds are provided:

wherein R2 is selected from the group consisting of Hydrogen, alkyl,substituted alkyl, and (CH₂)_(n) wherein n=1-6;

wherein R3 is selected from the group consisting of hydrogen, halogen,alkyl, substituted alkyl, carboxylic acid, amide SO₂NH₂, NHSO₂alkyl, andNO₂;

wherein X is selected from the group consisting of

alkyl, substituted alkyl, sulfolamide, SO₂alkyl, NHSO₂, CH₂, CH₂CH₂,SO₂, CH₂SO₂, SO₂CH₂, OCH₂CH₂O, SO, CH₂CH₂SO, SOCH₂CH₂; and

wherein L, M and N are present or absent, and are selected from thegroup consisting of alkyl, NO₂, halogen, OH, O-Alkyl, methyl ester,propyl ester, ethyl ester, CO₂H, CF₃, aniline, nitro, heterocycle,mono-substituted alkyl, di-substituted alkyl, and tri-substituted alkyl,hydrogen, SO₂NH₂, SO₂NH-alkyl, SOalkyl, NHSO₂alkyl; and

wherein Y is selected from the group consisting of hydrogen, alkyl,substituted alkyl, halogen, OH, O-Alkyl, methyl ester, propyl ester,ethyl ester, CO₂H, nitro, heterocycle, mono-substituted alkyl,di-substituted alkyl, and tri-substituted alkyl, hydrogen, SOalkyl,SO₂NH₂, SO₂NH-alkyl, NHSO₂alkyl, and

wherein WW, XX, YY and ZZ are present or absent, and are selected fromthe group consisting of alkyl, halogen, OH, O-Alkyl, methyl ester,propyl ester, ethyl ester, CO₂H, aniline, nitro, heterocycle,mono-substituted alkyl, di-substituted alkyl, and tri-substituted alkyl,hydrogen, SO₂NH₂, SO₂NH-alkyl, NHSO₂alkyl; and wherein Z is selectedfrom the group consisting of

wherein R5 is selected from the group consisting of alkyl,mono-substituted alkyl, di-substituted alkyl, and tri-substituted alkyl.

In some preferred embodiments, crystal forms and formulations of thefollowing exemplary compounds are provided:

In some preferred embodiments, crystal forms and formulations of thefollowing exemplary compound is provided:

In some preferred embodiments, crystal forms and formulations of thefollowing exemplary compounds are provided:

including both R and S enantiomeric forms and racemic mixtures; whereinR1 is selected from the group consisting of:

wherein X is selected from the group consisting of heteroatom, alkyl,and substituted alkly;

wherein Z and Y are separately selected from the group consisting of O,N and S;

wherein R2 is selected from the group consisting of methyl, H, alkyl,and (CH₂)_(n)-morpholino wherein n=1−6; and wherein R3 is selected fromthe group consisting of hydrogen, halogen, alkyl, substituted alkyl,carboxylic acid, amide SO₂NH₂, NHSO₂alkyl, and NO₂.

In some preferred embodiments, crystal forms and formulations of thefollowing exemplary compounds are provided:

wherein R1 is selected from the group consisting of methyl, hydrogen,alkyl, and (CH₂)_(n)-morpholino wherein n=1−6; wherein R2 is selectedfrom the group consisting of

wherein R3 is selected from the group consisting of hydrogen, halogen,alkyl, substituted alkyl, carboxylic acid, amide, SO₂NH₂, NHSO₂alkyl,and NO₂; wherein BB, CC, DD, and R4 are present or absent, and areselected from the group consisting of hydrogen, CF₃, NO₂, alkyl,halogen, OH, O-alkyl, nitro, OCH₂CH₂OH, SO₂H, mono-substituted alkyl,di-substituted alkyl, tri-substituted alkyl, CO₂H, heterocycle, SO₂NH₂,SO₂NH-alkyl, NHSO₂alkyl, methyl ester, propyl ester, and ethyl ester;and wherein R5 is selected from the group consisting of NHSO₂, CH₂NHSO₂,CH₂CH₂NHSO₂, CH₂CH₂CH₂NHSO₂, SO₂NH, SO₂NHCH₂, SO₂NHCH₂CH₂,SO₂NHCH₂CH₂CH₂, CH₂, CH₂CH₂, CH₂CH₂CH₂, SO₂, CH₂SO, SOCH₂, OCH₂CH₂O, SO,CH₂CH₂SO, and SOCH₂CH₂.

In some preferred embodiments, crystal forms and formulations of thefollowing exemplary compounds are provided:

wherein R₁ is selected from

In certain preferred embodiments, crystal forms and formulations of thefollowing exemplary compounds are provided:

including both R and S enantiomeric forms and racemic mixtures; whereinR1 is a nitrogen atom or a carbon atom; wherein R2 is comprises achemical moiety comprising a heterocyclic group containing 3 or morecarbon atoms; wherein R3 comprises a chemical moiety comprising aheterocyclic group containing 3 or more carbon atoms; and wherein R4 andR5 are separately selected from the group consisting of: hydrogen;halogen; CH₃; a linear or branched, saturated or unsaturated aliphaticchain having at least 2 carbons; a chemical moiety comprising a halogen;a chemical moiety comprising Sulfur; a chemical moiety comprisingNitrogen; an aromatic chemical moiety; a hydrophilic chemical moiety;and a hydrophobic chemical moiety.

In preferred embodiments, the compound comprises the formula:

wherein R6 is selected from the group consisting of H and a ketone; andwherein R7 is selected from the group consisting of H and a ketone.

In preferred embodiments, the compound comprises the formula:

In such preferred embodiments, R8 is carbon or nitrogen and R9 isselected from H, a hydroxy, an alkoxy, a halogen, an amino, alower-alkyl, a substituted-amino, an acetylamino, a hydroxyamino, analiphatic group having 1-8 carbons and 1-20 hydrogens, a substitutedaliphatic group of similar size, a cycloaliphatic group consisting ofless than 10 carbons, a substituted cycloaliphatic group, an aryl, aheterocyclic, NO₂; SR′; and NR′₂, wherein R′ is defined as a linear orbranched, saturated or unsaturated aliphatic chain having at least onecarbon; a linear or branched, saturated or unsaturated aliphatic chainhaving at least 2 carbons, and having at least one hydroxyl subgroup; alinear or branched, saturated or unsaturated aliphatic chain having atleast 2 carbons, and having at least one thiol subgroup; a linear orbranched, saturated or unsaturated aliphatic chain having at least 2carbons, wherein the aliphatic chain terminates with an aldehydesubgroup; a linear or branched, saturated or unsaturated aliphatic chainhaving at least 2 carbons, and having at least one ketone subgroup; alinear or branched, saturated or unsaturated aliphatic chain having atleast 2 carbons; wherein the aliphatic chain terminates with acarboxylic acid subgroup; a linear or branched, saturated or unsaturatedaliphatic chain having at least 2 carbons, and having at least one amidesubgroup; a linear or branched, saturated or unsaturated aliphatic chainhaving at least 2 carbons, and having at least one acyl group; a linearor branched, saturated or unsaturated aliphatic chain having at least 2carbons, and having at least one nitrogen containing moiety; a linear orbranched, saturated or unsaturated aliphatic chain having at least 2carbons, and having at least one amine subgroup; a linear or branched,saturated or unsaturated aliphatic chain having at least 2 carbons, andhaving at least one halogen subgroup; a linear or branched, saturated orunsaturated aliphatic chain having at least 2 carbons, and having atleast one nitronium subgroup.

In preferred embodiments, the compound comprises the formula:

wherein R9 is selected from H, a hydroxy, an alkoxy, a halo, an amino, alower-alkyl, a substituted-amino, an acetylamino, a hydroxyamino, analiphatic group having 1-8 carbons and 1-20 hydrogens, a substitutedaliphatic group of similar size, a cycloaliphatic group consisting ofless than 10 carbons, a substituted cycloaliphatic group, an aryl, aheterocyclic, NO₂; SR′; and NR′₂, wherein R′ is defined as a linear orbranched, saturated or unsaturated aliphatic chain having at least onecarbon; a linear or branched, saturated or unsaturated aliphatic chainhaving at least 2 carbons, and having at least one hydroxyl subgroup; alinear or branched, saturated or unsaturated aliphatic chain having atleast 2 carbons, and having at least one thiol subgroup; a linear orbranched, saturated or unsaturated aliphatic chain having at least 2carbons, wherein the aliphatic chain terminates with an aldehydesubgroup; a linear or branched, saturated or unsaturated aliphatic chainhaving at least 2 carbons, and having at least one ketone subgroup; alinear or branched, saturated or unsaturated aliphatic chain having atleast 2 carbons; wherein the aliphatic chain terminates with acarboxylic acid subgroup; a linear or branched, saturated or unsaturatedaliphatic chain having at least 2 carbons, and having at least one amidesubgroup; a linear or branched, saturated or unsaturated aliphatic chainhaving at least 2 carbons, and having at least one acyl group; a linearor branched, saturated or unsaturated aliphatic chain having at least 2carbons, and having at least one nitrogen containing moiety; a linear orbranched, saturated or unsaturated aliphatic chain having at least 2carbons, and having at least one amine subgroup; a linear or branched,saturated or unsaturated aliphatic chain having at least 2 carbons, andhaving at least one halogen subgroup; a linear or branched, saturated orunsaturated aliphatic chain having at least 2 carbons, and having atleast one nitronium subgroup.

In preferred embodiments, the compound comprises the formula:

wherein R10 is selected from the group consisting of: hydrogen; halogen;CH₃; a linear or branched, saturated or unsaturated aliphatic chainhaving at least 2 carbons; a chemical moiety comprising a halogen; achemical moiety comprising Sulfur; a chemical moiety comprisingNitrogen; an aromatic chemical moiety; a hydrophilic chemical moiety;and a hydrophobic chemical moiety; and wherein R7 is selected from thegroup consisting of H and a ketone.

In other preferred embodiments, R3 is selected from the group consistingof:

wherein R12, R13, R14 and R15 are selected from the group consisting of:hydrogen; CH₃; a linear or branched, saturated or unsaturated aliphaticchain having at least 1 carbon; a linear or branched, saturated orunsaturated aliphatic chain having at least 2 carbons, and having atleast one hydroxy subgroup; a linear or branched, saturated orunsaturated aliphatic chain having at least 2 carbons, and having atleast one thiol subgroup; a linear or branched, saturated or unsaturatedaliphatic chain having at least 2 carbons, wherein the aliphatic chainterminates with an aldehyde subgroup; a linear or branched, saturated orunsaturated aliphatic chain having at least 2 carbons, and having atleast one ketone subgroup; a linear or branched, saturated orunsaturated aliphatic chain having at least 2 carbons; wherein thealiphatic chain terminates with a carboxylic acid subgroup; a linear orbranched, saturated or unsaturated aliphatic chain having at least 2carbons, and having at least one amide subgroup; a linear or branched,saturated or unsaturated aliphatic chain having at least 2 carbons, andhaving at least one acyl group; a linear or branched, saturated orunsaturated aliphatic chain having at least 2 carbons, and having atleast one nitrogen containing moiety; a linear or branched, saturated orunsaturated aliphatic chain having at least 2 carbons, and having atleast one amine subgroup; a linear or branched, saturated or unsaturatedaliphatic chain having at least 2 carbons, and having at least one ethersubgroup; a linear or branched, saturated or unsaturated aliphatic chainhaving at least 2 carbons, and having at least one halogen subgroup; alinear or branched, saturated or unsaturated aliphatic chain having atleast 2 carbons, and having at least one nitronium subgroup; and R11 isOH.

In yet other preferred embodiments, R4 or R5 are selected from groupconsisting of: napthalalanine; phenol; 1-Napthalenol; 2-Napthalenol;

quinolines, and all aromatic regioisomers.

In other preferred embodiments, R4 or R5 is selected from the groupconsisting of:

wherein R16 is carbon or nitrogen; wherein R17 is selected from thegroup consisting of hydrogen; halogen; CH₃; a linear or branched,saturated or unsaturated aliphatic chain having at least 2 carbons; achemical moiety comprising a halogen; a chemical moiety comprisingSulfur; a chemical moiety comprising Nitrogen; an aromatic chemicalmoiety; a hydrophilic chemical moiety; and a hydrophobic chemicalmoiety; wherein R18 is carbon or nitrogen; wherein R19 is selected fromthe group consisting of hydrogen; halogen; CH₃; a linear or branched,saturated or unsaturated aliphatic chain having at least 2 carbons; achemical moiety comprising a halogen; a chemical moiety comprisingSulfur; a chemical moiety comprising Nitrogen; an aromatic chemicalmoiety; a hydrophilic chemical moiety; and a hydrophobic chemicalmoiety; and wherein R20 is carbon or nitrogen.

In some preferred embodiments, crystal forms and formulations of thefollowing exemplary compounds are provided:

including both R and S enantiomeric forms and racemic mixtures. In suchpreferred embodiments, R1 is a nitrogen atom or a carbon atom; R2 iscarbon or nitrogen; R3 comprises a chemical moiety comprising aheterocyclic group containing 3 or more carbon atoms; R4 and R5 areseparately selected from the group consisting of: hydrogen; halogen;CH₃; a linear or branched, saturated or unsaturated aliphatic chainhaving at least 2 carbons; a chemical moiety comprising a halogen; achemical moiety comprising Sulfur; a chemical moiety comprisingNitrogen; an aromatic chemical moiety; a hydrophilic chemical moiety;and a hydrophobic chemical moiety; and R6 is selected from H, a hydroxy,an alkoxy, a halogen, an amino, a lower-alkyl, a substituted-amino, anacetylamino, a hydroxyamino, an aliphatic group having 1-8 carbons and1-20 hydrogens, a substituted aliphatic group of similar size, acycloaliphatic group consisting of less than 10 carbons, a substitutedcycloaliphatic group, an aryl, a heterocyclic, NO₂; SR′; and NR′₂,wherein R′ is defined as a linear or branched, saturated or unsaturatedaliphatic chain having at least one carbon; a linear or branched,saturated or unsaturated aliphatic chain having at least 2 carbons, andhaving at least one hydroxyl subgroup; a linear or branched, saturatedor unsaturated aliphatic chain having at least 2 carbons, and having atleast one thiol subgroup; a linear or branched, saturated or unsaturatedaliphatic chain having at least 2 carbons, wherein the aliphatic chainterminates with an aldehyde subgroup; a linear or branched, saturated orunsaturated aliphatic chain having at least 2 carbons, and having atleast one ketone subgroup; a linear or branched, saturated orunsaturated aliphatic chain having at least 2 carbons; wherein thealiphatic chain terminates with a carboxylic acid subgroup; a linear orbranched, saturated or unsaturated aliphatic chain having at least 2carbons, and having at least one amide subgroup; a linear or branched,saturated or unsaturated aliphatic chain having at least 2 carbons, andhaving at least one acyl group; a linear or branched, saturated orunsaturated aliphatic chain having at least 2 carbons, and having atleast one nitrogen containing moiety; a linear or branched, saturated orunsaturated aliphatic chain having at least 2 carbons, and having atleast one amine subgroup; a linear or branched, saturated or unsaturatedaliphatic chain having at least 2 carbons, and having at least onehalogen subgroup; a linear or branched, saturated or unsaturatedaliphatic chain having at least 2 carbons, and having at least onenitronium subgroup.

In preferred embodiments, R3 is selected from the group consisting of:

wherein R12, R13, R14 and R15 are selected from the group consisting of:hydrogen; CH₃; a linear or branched, saturated or unsaturated aliphaticchain having at least 1 carbon; a linear or branched, saturated orunsaturated aliphatic chain having at least 2 carbons, and having atleast one hydroxy subgroup; a linear or branched, saturated orunsaturated aliphatic chain having at least 2 carbons, and having atleast one thiol subgroup; a linear or branched, saturated or unsaturatedaliphatic chain having at least 2 carbons, wherein the aliphatic chainterminates with an aldehyde subgroup; a linear or branched, saturated orunsaturated aliphatic chain having at least 2 carbons, and having atleast one ketone subgroup; a linear or branched, saturated orunsaturated aliphatic chain having at least 2 carbons; wherein thealiphatic chain terminates with a carboxylic acid subgroup; a linear orbranched, saturated or unsaturated aliphatic chain having at least 2carbons, and having at least one amide subgroup; a linear or branched,saturated or unsaturated aliphatic chain having at least 2 carbons, andhaving at least one acyl group; a linear or branched, saturated orunsaturated aliphatic chain having at least 2 carbons, and having atleast one nitrogen containing moiety; a linear or branched, saturated orunsaturated aliphatic chain having at least 2 carbons, and having atleast one amine subgroup; a linear or branched, saturated or unsaturatedaliphatic chain having at least 2 carbons, and having at least one ethersubgroup; a linear or branched, saturated or unsaturated aliphatic chainhaving at least 2 carbons, and having at least one halogen subgroup; alinear or branched, saturated or unsaturated aliphatic chain having atleast 2 carbons, and having at least one nitronium subgroup; and R11 isOH.

In preferred embodiments, R4 or R5 are selected from group consistingof:

quinolines, and all aromatic regioisomers.

In other preferred embodiments, R4 or R5 is selected from the groupconsisting of:

wherein R16 is carbon or nitrogen; wherein R17 is selected from thegroup consisting of hydrogen; halogen; CH₃; a linear or branched,saturated or unsaturated aliphatic chain having at least 2 carbons; achemical moiety comprising a halogen; a chemical moiety comprisingSulfur; a chemical moiety comprising Nitrogen; an aromatic chemicalmoiety; a hydrophilic chemical moiety; and a hydrophobic chemicalmoiety; wherein R18 is carbon or nitrogen; wherein R19 is selected fromthe group consisting of hydrogen; halogen; CH₃; a linear or branched,saturated or unsaturated aliphatic chain having at least 2 carbons; achemical moiety comprising a halogen; a chemical moiety comprisingSulfur; a chemical moiety comprising Nitrogen; an aromatic chemicalmoiety; a hydrophilic chemical moiety; and a hydrophobic chemicalmoiety; and wherein R20 is carbon or nitrogen.

In some preferred embodiments, crystal forms and formulations of thefollowing exemplary compounds are provided:

In some preferred embodiments, crystal forms and formulations of thefollowing exemplary compounds are provided:

including both R and S enantiomeric forms and racemic mixtures.

In such preferred embodiments, R1 is a nitrogen atom or a carbon atom;R2 is selected from H, a hydroxy, an alkoxy, a halo, an amino, alower-alkyl, a substituted-amino, an acetylamino, a hydroxyamino, analiphatic group having 1-8 carbons and 1-20 hydrogens, a substitutedaliphatic group of similar size, a cycloaliphatic group consisting ofless than 10 carbons, a substituted cycloaliphatic group, an aryl, aheterocyclic, NO₂; SR′; and NR′₂, wherein R′ is defined as a linear orbranched, saturated or unsaturated aliphatic chain having at least onecarbon; a linear or branched, saturated or unsaturated aliphatic chainhaving at least 2 carbons, and having at least one hydroxyl subgroup; alinear or branched, saturated or unsaturated aliphatic chain having atleast 2 carbons, and having at least one thiol subgroup; a linear orbranched, saturated or unsaturated aliphatic chain having at least 2carbons, wherein the aliphatic chain terminates with an aldehydesubgroup; a linear or branched, saturated or unsaturated aliphatic chainhaving at least 2 carbons, and having at least one ketone subgroup; alinear or branched, saturated or unsaturated aliphatic chain having atleast 2 carbons; wherein the aliphatic chain terminates with acarboxylic acid subgroup; a linear or branched, saturated or unsaturatedaliphatic chain having at least 2 carbons, and having at least one amidesubgroup; a linear or branched, saturated or unsaturated aliphatic chainhaving at least 2 carbons, and having at least one acyl group; a linearor branched, saturated or unsaturated aliphatic chain having at least 2carbons, and having at least one nitrogen containing moiety; a linear orbranched, saturated or unsaturated aliphatic chain having at least 2carbons, and having at least one amine subgroup; a linear or branched,saturated or unsaturated aliphatic chain having at least 2 carbons, andhaving at least one halogen subgroup; a linear or branched, saturated orunsaturated aliphatic chain having at least 2 carbons, and having atleast one nitronium subgroup; R3 comprises a chemical moiety comprisinga heterocyclic group containing 3 or more carbon atoms; and R4 and R5are separately selected from the group consisting of: hydrogen; halogen;CH₃; a linear or branched, saturated or unsaturated aliphatic chainhaving at least 2 carbons; a chemical moiety comprising a halogen; achemical moiety comprising Sulfur; a chemical moiety comprisingNitrogen; an aromatic chemical moiety; a hydrophilic chemical moiety;and a hydrophobic chemical moiety.

In preferred embodiments, R3 is selected from the group consisting of:

wherein R12, R13, R14 and R15 are selected from the group consisting of:hydrogen; CH₃; a linear or branched, saturated or unsaturated aliphaticchain having at least 1 carbon; a linear or branched, saturated orunsaturated aliphatic chain having at least 2 carbons, and having atleast one hydroxy subgroup; a linear or branched, saturated orunsaturated aliphatic chain having at least 2 carbons, and having atleast one thiol subgroup; a linear or branched, saturated or unsaturatedaliphatic chain having at least 2 carbons, wherein the aliphatic chainterminates with an aldehyde subgroup; a linear or branched, saturated orunsaturated aliphatic chain having at least 2 carbons, and having atleast one ketone subgroup; a linear or branched, saturated orunsaturated aliphatic chain having at least 2 carbons; wherein thealiphatic chain terminates with a carboxylic acid subgroup; a linear orbranched, saturated or unsaturated aliphatic chain having at least 2carbons, and having at least one amide subgroup; a linear or branched,saturated or unsaturated aliphatic chain having at least 2 carbons, andhaving at least one acyl group; a linear or branched, saturated orunsaturated aliphatic chain having at least 2 carbons, and having atleast one nitrogen containing moiety; a linear or branched, saturated orunsaturated aliphatic chain having at least 2 carbons, and having atleast one amine subgroup; a linear or branched, saturated or unsaturatedaliphatic chain having at least 2 carbons, and having at least one ethersubgroup; a linear or branched, saturated or unsaturated aliphatic chainhaving at least 2 carbons, and having at least one halogen subgroup; alinear or branched, saturated or unsaturated aliphatic chain having atleast 2 carbons, and having at least one nitronium subgroup; and R11 isOH.

In preferred embodiments, R4 or R5 are selected from group consistingof:

quinolines, and all aromatic regioisomers.

In other preferred embodiments, R4 or R5 is selected from the groupconsisting of:

wherein R16 is carbon or nitrogen; wherein R17 is selected from thegroup consisting of hydrogen; halogen; CH₃; a linear or branched,saturated or unsaturated aliphatic chain having at least 2 carbons; achemical moiety comprising a halogen; a chemical moiety comprisingSulfur; a chemical moiety comprising Nitrogen; an aromatic chemicalmoiety; a hydrophilic chemical moiety; and a hydrophobic chemicalmoiety; wherein R18 is carbon or nitrogen; wherein R19 is selected fromthe group consisting of hydrogen; halogen; CH₃; a linear or branched,saturated or unsaturated aliphatic chain having at least 2 carbons; achemical moiety comprising a halogen; a chemical moiety comprisingSulfur; a chemical moiety comprising Nitrogen; an aromatic chemicalmoiety; a hydrophilic chemical moiety; and a hydrophobic chemicalmoiety; and wherein R20 is carbon or nitrogen.

In some preferred embodiments, crystal forms and formulations of thefollowing exemplary compounds are provided:

including both R and S enantiomeric forms and racemic mixtures.

In such preferred embodiments, R1 is carbon or nitrogen; R2 is selectedfrom H, a hydroxy, an alkoxy, a halogen, an amino, a lower-alkyl, asubstituted-amino, an acetylamino, a hydroxyamino, an aliphatic grouphaving 1-8 carbons and 1-20 hydrogens, a substituted aliphatic group ofsimilar size, a cycloaliphatic group consisting of less than 10 carbons,a substituted cycloaliphatic group, an aryl, a heterocyclic, NO₂; SR′;and NR′₂, wherein R′ is defined as a linear or branched, saturated orunsaturated aliphatic chain having at least one carbon; a linear orbranched, saturated or unsaturated aliphatic chain having at least 2carbons, and having at least one hydroxyl subgroup; a linear orbranched, saturated or unsaturated aliphatic chain having at least 2carbons, and having at least one thiol subgroup; a linear or branched,saturated or unsaturated aliphatic chain having at least 2 carbons,wherein the aliphatic chain terminates with an aldehyde subgroup; alinear or branched, saturated or unsaturated aliphatic chain having atleast 2 carbons, and having at least one ketone subgroup; a linear orbranched, saturated or unsaturated aliphatic chain having at least 2carbons; wherein the aliphatic chain terminates with a carboxylic acidsubgroup; a linear or branched, saturated or unsaturated aliphatic chainhaving at least 2 carbons, and having at least one amide subgroup; alinear or branched, saturated or unsaturated aliphatic chain having atleast 2 carbons, and having at least one acyl group; a linear orbranched, saturated or unsaturated aliphatic chain having at least 2carbons, and having at least one nitrogen containing moiety; a linear orbranched, saturated or unsaturated aliphatic chain having at least 2carbons, and having at least one amine subgroup; a linear or branched,saturated or unsaturated aliphatic chain having at least 2 carbons, andhaving at least one halogen subgroup; a linear or branched, saturated orunsaturated aliphatic chain having at least 2 carbons, and having atleast one nitronium subgroup; R3 comprises a chemical moiety comprisinga heterocyclic group containing 3 or more carbon atoms; R4 is selectedfrom the group consisting of: hydrogen; halogen; CH₃; a linear orbranched, saturated or unsaturated aliphatic chain having at least 2carbons; a chemical moiety comprising a halogen; a chemical moietycomprising Sulfur; a chemical moiety comprising Nitrogen; an aromaticchemical moiety; a hydrophilic chemical moiety; and a hydrophobicchemical moiety; and R5 is selected from the group consisting of:hydrogen; halogen; CH₃; a linear or branched, saturated or unsaturatedaliphatic chain having at least 2 carbons; a chemical moiety comprisinga halogen; a chemical moiety comprising Sulfur; a chemical moietycomprising Nitrogen; an aromatic chemical moiety; a hydrophilic chemicalmoiety; and a hydrophobic chemical moiety.

In preferred embodiments, R3 is selected from the group consisting of:

wherein R12, R13, R14 and R15 are selected from the group consisting of:hydrogen; CH₃; a linear or branched, saturated or unsaturated aliphaticchain having at least 1 carbon; a linear or branched, saturated orunsaturated aliphatic chain having at least 2 carbons, and having atleast one hydroxy subgroup; a linear or branched, saturated orunsaturated aliphatic chain having at least 2 carbons, and having atleast one thiol subgroup; a linear or branched, saturated or unsaturatedaliphatic chain having at least 2 carbons, wherein the aliphatic chainterminates with an aldehyde subgroup; a linear or branched, saturated orunsaturated aliphatic chain having at least 2 carbons, and having atleast one ketone subgroup; a linear or branched, saturated orunsaturated aliphatic chain having at least 2 carbons; wherein thealiphatic chain terminates with a carboxylic acid subgroup; a linear orbranched, saturated or unsaturated aliphatic chain having at least 2carbons, and having at least one amide subgroup; a linear or branched,saturated or unsaturated aliphatic chain having at least 2 carbons, andhaving at least one acyl group; a linear or branched, saturated orunsaturated aliphatic chain having at least 2 carbons, and having atleast one nitrogen containing moiety; a linear or branched, saturated orunsaturated aliphatic chain having at least 2 carbons, and having atleast one amine subgroup; a linear or branched, saturated or unsaturatedaliphatic chain having at least 2 carbons, and having at least one ethersubgroup; a linear or branched, saturated or unsaturated aliphatic chainhaving at least 2 carbons, and having at least one halogen subgroup; alinear or branched, saturated or unsaturated aliphatic chain having atleast 2 carbons, and having at least one nitronium subgroup; and R11 isOH.

In preferred embodiments, R4 or R5 are selected from group consistingof:

quinolines, and all aromatic regioisomers.

In other preferred embodiments, R4 or R5 is selected from the groupconsisting of:

wherein R16 is carbon or nitrogen; wherein R17 is selected from thegroup consisting of hydrogen; halogen; CH₃; a linear or branched,saturated or unsaturated aliphatic chain having at least 2 carbons; achemical moiety comprising a halogen; a chemical moiety comprisingSulfur; a chemical moiety comprising Nitrogen; an aromatic chemicalmoiety; a hydrophilic chemical moiety; and a hydrophobic chemicalmoiety; wherein R18 is carbon or nitrogen; wherein R19 is selected fromthe group consisting of hydrogen; halogen; CH₃; a linear or branched,saturated or unsaturated aliphatic chain having at least 2 carbons; achemical moiety comprising a halogen; a chemical moiety comprisingSulfur; a chemical moiety comprising Nitrogen; an aromatic chemicalmoiety; a hydrophilic chemical moiety; and a hydrophobic chemicalmoiety; and wherein R20 is carbon or nitrogen.

In certain embodiments, crystal forms and formulations of the followingexemplary compounds comprising the following formula are provided:A-B-C; wherein A is a chemical moiety comprising a hydroxyl group (e.g.,a phenolic ring); wherein B is a chemical moiety (e.g., scaffoldmolecule) separating A and C by at least 1 atom; and wherein C is ahydrophobic chemical moiety (e.g., naphyl group).

In some embodiments, A is selected from the group consisting of: isselected from the group consisting of:

wherein R1′, R2, R3 and R4 are selected from the group consisting of:hydrogen; CH₃; a linear or branched, saturated or unsaturated aliphaticchain having at least 1 carbon; a linear or branched, saturated orunsaturated aliphatic chain having at least 2 carbons, and having atleast one hydroxy subgroup; a linear or branched, saturated orunsaturated aliphatic chain having at least 2 carbons, and having atleast one thiol subgroup; a linear or branched, saturated or unsaturatedaliphatic chain having at least 2 carbons, wherein the aliphatic chainterminates with an aldehyde subgroup; a linear or branched, saturated orunsaturated aliphatic chain having at least 2 carbons, and having atleast one ketone subgroup; a linear or branched, saturated orunsaturated aliphatic chain having at least 2 carbons; wherein thealiphatic chain terminates with a carboxylic acid subgroup; a linear orbranched, saturated or unsaturated aliphatic chain having at least 2carbons, and having at least one amide subgroup; a linear or branched,saturated or unsaturated aliphatic chain having at least 2 carbons, andhaving at least one acyl group; a linear or branched, saturated orunsaturated aliphatic chain having at least 2 carbons, and having atleast one nitrogen containing moiety; a linear or branched, saturated orunsaturated aliphatic chain having at least 2 carbons, and having atleast one amine subgroup; a linear or branched, saturated or unsaturatedaliphatic chain having at least 2 carbons, and having at least one ethersubgroup; a linear or branched, saturated or unsaturated aliphatic chainhaving at least 2 carbons, and having at least one halogen subgroup; alinear or branched, saturated or unsaturated aliphatic chain having atleast 2 carbons, and having at least one nitronium subgroup; and R5 isOH.

In some embodiments, C is selected from group consisting of:napthalalanine; phenol; 1-Napthalenol; 2-Napthalenol;

and aromatic regioisomers.

In some embodiments, C comprises an aryl group and/or an aliphaticgroup.

In some embodiments, B is a benzodiazepine structure described by thefollowing formula:

In some embodiments, A is located at position 5 of the benzodiazepinestructure. In some preferred embodiments, C is located at position 3 ofthe benzodiazepine structure. In other preferred embodiments, A islocated at a position of the benzodiazepine structure selected from thegroup consisting of position 1, position 2, position 3, position 4,position 5, position 6, position 7, position 8, position 9, and position10.

In some embodiments, crystal forms and formulations of the followingexemplary compounds are provided:

Certain embodiments of the present invention include crystal forms andformulations of exemplary compounds with the following formula:

including both R and S enantiomeric forms and racemic mixtures, whereinR1 is an electron rich heterocycle.

In preferred embodiments, R1 is selected from the group consisting of:

In some embodiments, R2 is a halogen. In some embodiments, R2 isChlorine.

In certain embodiments, examples of crystal forms and formulations ofthe exemplary 1,4-benzodiazepine-2,5-dione compounds compounds includebut are not limited to:

Certain embodiments of the present invention include crystal forms andformulations of benzodiazepine (and benzodiazepine related) compoundshaving a chemical moiety that causes the benzodiazepine to lack a chiralcenter associated with the third carbon position of the benzodiazepinering. Certain embodiments of the present invention include crystal formsand formulations of exemplary compounds with the following formula:

including both R and S enantiomeric forms and racemic mixtures.

In some embodiments, A - - - B is selected from the group consisting ofN—CH₂, and C═N.

In some embodiments, R1 is selected from the group consisting of

R1′ is selected from the group consisting of halogen; alkyl; substitutedalkyl; aryl; substituted aryl; amino; carbonyl; sulfone; sulfonamide;ether; OH; a chemical moiety comprising Sulfur; a chemical moietycomprising Nitrogen; CH₃; a linear or branched, saturated or unsaturatedaliphatic chain having at least 1 carbon; a linear or branched,saturated or unsaturated aliphatic chain having at least 2 carbons, andhaving at least one hydroxy subgroup; a linear or branched, saturated orunsaturated aliphatic chain having at least 2 carbons, and having atleast one thiol subgroup; a linear or branched, saturated or unsaturatedaliphatic chain having at least 2 carbons, wherein said aliphatic chainterminates with an aldehyde subgroup; a linear or branched, saturated orunsaturated aliphatic chain having at least 2 carbons, and having atleast one ketone subgroup; a linear or branched, saturated orunsaturated aliphatic chain having at least 2 carbons; wherein saidaliphatic chain terminates with a carboxylic acid subgroup; a linear orbranched, saturated or unsaturated aliphatic chain having at least 2carbons, and having at least one amide subgroup; a linear or branched,saturated or unsaturated aliphatic chain having at least 2 carbons, andhaving at least one acyl group; a linear or branched, saturated orunsaturated aliphatic chain having at least 2 carbons, and having atleast one nitrogen containing moiety; a linear or branched, saturated orunsaturated aliphatic chain having at least 2 carbons, and having atleast one amine subgroup; a linear or branched, saturated or unsaturatedaliphatic chain having at least 2 carbons, and having at least one ethersubgroup; a linear or branched, saturated or unsaturated aliphatic chainhaving at least 2 carbons, and having at least one halogen subgroup; alinear or branched, saturated or unsaturated aliphatic chain having atleast 2 carbons, and having at least one nitronium subgroup.

In some embodiments, R2 is an aliphatic cyclic group larger thanbenzene, wherein said larger than benzene comprises any chemical groupcontaining 7 or more non-hydrogen atoms.

In some embodiments, R2 is selected from group consisting of:napthalalanine; phenol; 1-Napthalenol; 2-Napthalenol;

quinolines, and all aromatic regioisomers.

In some embodiments, R2 is:

wherein X is selected from the group consisting of

alkyl, substituted alkyl, sulfolamide, SO₂alkyl, NHSO₂, CH₂, CH₂CH₂,SO₂, CH₂SO₂, SO₂CH₂, OCH₂CH₂O, SO, CH₂CH₂SO, SOCH₂CH₂; and wherein L, Mand N are present or absent, and are selected from the group consistingof alkyl, NO₂, halogen, OH, O-Alkyl, methyl ester, propyl ester, ethylester, CO₂H, CF₃, aniline, nitro, heterocycle, mono-substituted alkyl,di-substituted alkyl, and tri-substituted alkyl, hydrogen, SO₂NH₂,SO₂NH-alkyl, SOalkyl, NHSO₂alkyl; wherein Y is selected from the groupconsisting of hydrogen, alkyl, substituted alkyl, halogen, OH, O-Alkyl,methyl ester, propyl ester, ethyl ester, CO₂H, nitro, heterocycle,mono-substituted alkyl, di-substituted alkyl, and tri-substituted alkyl,hydrogen, SOalkyl, SO₂NH2, SO₂NH-alkyl, NHSO₂alkyl, and

wherein WW, XX, YY and ZZ are present or absent, and are selected fromthe group consisting of alkyl, halogen, OH, O-Alkyl, methyl ester,propyl ester, ethyl ester, CO₂H, aniline, nitro, heterocycle,mono-substituted alkyl, di-substituted alkyl, and tri-substituted alkyl,hydrogen, SO₂NH₂, SO₂NH-alkyl, and NHSO₂alkyl.

In some embodiments, R3 is an isostere of OH. In some embodiments, R3 isselected from the group consisting of hydrogen; halogen; OH; MnO4; alinear or branched, saturated or unsaturated, substituted ornon-substituted, aliphatic chain having at least 2 carbons; a chemicalmoiety comprising Sulfur; a chemical moiety comprising Nitrogen. In someembodiments, R3 is selected from the group consisting of alkyl;mono-substituted alkyl; di-substituted alkyl; tri-substituted alkyl;(CH₂)_(n) wherein n=1−6; CN; N₃; CNO; NH₂; SH; CF₃; OCH₃;NCH₂CH(CH₂)N(CH₃)₂; NCH₂CHCH₂N(CH₃)₂; phenyl; 2-pyridyl; 3-pyridyl;4-pyridyl; NCH₃; NCONHCH₃; CH₂OH; NHCONH₂; NHCOCH₃; NHSO₂CH₃; NHCN;NHCHO; SOCH₃; SO₂CH₃; CHNOH; CHNOCH₃; SCH₃; CH₂CO; CH₂SO₂; CONH;CH₂C(NOH); CH₂C(NOMe); NHSO₂PH; NHCS; CH₂NHCO; COCH₂; NHCO₂; and NHCOS.In some embodiments, R3 is described by any of the isosteres describedin, for example, Patani, G. and LaVoie, E. J., 1996, Chem. Rev.96:3147-3176; herein incorporated by reference in its entirety.

In some embodiments, R4 is a chemical moiety that causes thebenzodiazepine to lack a chiral center. In some embodiments, R4 ishydrogen,

wherein R4′ is a linear or branched, saturated or unsaturated,substituted or non-substituted, aliphatic chain having at least 2carbons.

Certain embodiments of the present invention include crystal forms andformulations of exemplary compounds with the following formula:

In some embodiments, the present invention includes crystal forms andformulations of the following exemplary compound:

Additional exemplary compounds and uses useful in the present inventionare described at U.S. Patent Publication 2004/0009972, published Jan.15, 2004, herein incorporated by reference in its entirety. Additionalexemplary compounds and uses useful in the present invention aredescribed in U.S. Provisional Patent Nos. 60/131,761, 60/165,511,60/191,855, 60/312,560, 60/313,689, 60/396,670, 60/565,788, 60/607,599,60/641,040, and U.S. patent application Ser. Nos. 11/324,419,11/176,719, 11/110,228, 10/935,333, 10/886,450, 10/795,535, 10/634,114,10/427,211, 10/427,212, 10/217,878, 09/767,283, 09/700,101, and relatedapplications; each herein incorporated by reference in their entireties.Additional exemplary compounds and uses useful in the present inventionare described in Atwal, et al., Bioorg. Med. Chem. Lett. 14, 1027-1030(2004) and Atwal, et al., J. Med. Chem. 47, 1081-1084 (2004); eachherein incorporated by reference in their entireties.

Further, it should be understood that the numerical ranges giventhroughout this disclosure should be construed as a flexible range thatcontemplates any possible subrange within that range. For example, thedescription of a group having the range of 1-10 carbons would alsocontemplate a group possessing a subrange of, for example, 1-3, 1-5,1-8, or 2-3, 2-5, 2-8, 3-4, 3-5, 3-7, 3-9, 3-10, etc., carbons. Thus,the range 1-10 should be understood to represent the outer boundaries ofthe range within which many possible subranges are clearly contemplated.Additional examples contemplating ranges in other contexts can be foundthroughout this disclosure wherein such ranges include analogoussubranges within.

In summary, a large number of compounds are presented herein. Any one ormore of these compounds can be used to treat a variety of dysregulatorydisorders related to cellular death as described elsewhere herein.Additionally, any one or more of these compounds can be used to inhibitATP Hydrolysis while not affecting cell synthesis or cell viability.Additionally, any one or more of these compounds can be used incombination with at least one other therapeutic agent (e.g., potassiumchannel openers, calcium channel blockers, sodium hydrogen exchangerinhibitors, antiarrhythmic agents, antiatherosclerotic agents,anticoagulants, antithrombotic agents, prothrombolytic agents,fibrinogen antagonists, diuretics, antihypertensive agents, ATPaseinhibitors, mineralocorticoid receptor antagonists, phospodiesteraseinhibitors, antidiabetic agents, anti-inflammatory agents, antioxidants,angiogenesis modulators, antiosteoporosis agents, hormone replacementtherapies, hormone receptor modulators, oral contraceptives, antiobesityagents, antidepressants, antianxiety agents, antipsychotic agents,antiproliferative agents, antitumor agents, antiulcer andgastroesophageal reflux disease agents, growth hormone agents and/orgrowth hormone secretagogues, thyroid mimetics, anti-infective agents,antiviral agents, antibacterial agents, antifungal agents,cholesterol/lipid lowering agents and lipid profile therapies, andagents that mimic ischemic preconditioning and/or myocardial stunning,antiatherosclerotic agents, anticoagulants, antithrombotic agents,antihypertensive agents, antidiabetic agents, and antihypertensiveagents selected from ACE inhibitors, AT-1 receptor antagonists, ETreceptor antagonists, dual ET/AII receptor antagonists, andvasopepsidase inhibitors, or an antiplatelet agent selected fromGPIIb/IIIa blockers, P2Y₁ and P2Y₁₂ antagonists, thromboxane receptorantagonists, and aspirin) in along with a pharmaceutically-acceptablecarrier or diluent in a pharmaceutical composition. Additionally, anyone or more of these compounds can be used to treat a mitochondrial F₁F₀ATP hydrolase associated disorder (e.g., myocardial infarction,ventricular hypertrophy, coronary artery disease, non-Q wave MI,congestive heart failure, cardiac arrhythmias, unstable angina, chronicstable angina, Prinzmetal's angina, high blood pressure, intermittentclaudication, peripheral occlusive arterial disease, thrombotic orthromboembolic symptoms of thromboembolic stroke, venous thrombosis,arterial thrombosis, cerebral thrombosis, pulmonary embolism, cerebralembolism, thrombophilia, disseminated intravascular coagulation,restenosis, atrial fibrillation, ventricular enlargement,atherosclerotic vascular disease, atherosclerotic plaque rupture,atherosclerotic plaque formation, transplant atherosclerosis, vascularremodeling atherosclerosis, cancer, surgery, inflammation, systematicinfection, artificial surfaces, interventional cardiology, immobility,medication, pregnancy and fetal loss, and diabetic complicationscomprising retinopathy, nephropathy and neuropathy) in a patient. Theabove-described compounds can also be used in drug screening assays andother diagnostic methods.

IV. Pharmaceutical Compositions, Formulations, and ExemplaryAdministration Routes and Dosing Considerations

Exemplary embodiments of various contemplated medicaments andpharmaceutical compositions are provided below.

A. Preparing Medicaments

The compounds of the present invention are useful in the preparation ofmedicaments to treat a variety of conditions associated withdysregulation of cell death, aberrant cell growth andhyperproliferation.

In addition, the compounds are also useful for preparing medicaments fortreating other disorders wherein the effectiveness of the compounds areknown or predicted. Such disorders include, but are not limited to,neurological (e.g., epilepsy) or neuromuscular disorders. The methodsand techniques for preparing medicaments of a compound are well-known inthe art. Exemplary pharmaceutical formulations and routes of deliveryare described below.

One of skill in the art will appreciate that any one or more of thecompounds described herein, including the many specific embodiments, areprepared by applying standard pharmaceutical manufacturing procedures.Such medicaments can be delivered to the subject by using deliverymethods that are well-known in the pharmaceutical arts.

B. Exemplary Pharmaceutical Compositions and Formulation

In some embodiments of the present invention, the compositions areadministered alone, while in some other embodiments, the compositionsare preferably present in a pharmaceutical formulation comprising atleast one active ingredient/agent (e.g., benzodiazepine crystal formsand formulations and benzodiazepine related crystal forms andformulations), as defined above, together with a solid support oralternatively, together with one or more pharmaceutically acceptablecarriers and optionally other therapeutic agents. Each carrier should be“acceptable” in the sense that it is compatible with the otheringredients of the formulation and not injurious to the subject.

Contemplated formulations include those suitable oral, rectal, nasal,topical (including transdermal, buccal and sublingual), vaginal,parenteral (including subcutaneous, intramuscular, intravenous andintradermal) and pulmonary administration. In some embodiments,formulations are conveniently presented in unit dosage form and areprepared by any method known in the art of pharmacy. Such methodsinclude the step of bringing into association the active ingredient withthe carrier which constitutes one or more accessory ingredients. Ingeneral, the formulations are prepared by uniformly and intimatelybringing into association (e.g., mixing) the active ingredient withliquid carriers or finely divided solid carriers or both, and then ifnecessary shaping the product.

Formulations of the present invention suitable for oral administrationmay be presented as discrete units such as capsules, cachets or tablets,wherein each preferably contains a predetermined amount of the activeingredient; as a powder or granules; as a solution or suspension in anaqueous or non-aqueous liquid; or as an oil-in-water liquid emulsion ora water-in-oil liquid emulsion. In other embodiments, the activeingredient is presented as a bolus, electuary, or paste, etc.

In some embodiments, tablets comprise at least one active ingredient andoptionally one or more accessory agents/carriers are made by compressingor molding the respective agents. In preferred embodiments, compressedtablets are prepared by compressing in a suitable machine the activeingredient in a free-flowing form such as a powder or granules,optionally mixed with a binder (e.g., povidone, gelatin,hydroxypropylmethyl cellulose), lubricant, inert diluent, preservative,disintegrant (e.g., sodium starch glycolate, cross-linked povidone,cross-linked sodium carboxymethyl cellulose)surface-active or dispersingagent. Molded tablets are made by molding in a suitable machine amixture of the powdered compound (e.g., active ingredient) moistenedwith an inert liquid diluent. Tablets may optionally be coated or scoredand may be formulated so as to provide slow or controlled release of theactive ingredient therein using, for example, hydroxypropylmethylcellulose in varying proportions to provide the desired release profile.Tablets may optionally be provided with an enteric coating, to providerelease in parts of the gut other than the stomach.

Formulations suitable for topical administration in the mouth includelozenges comprising the active ingredient in a flavored basis, usuallysucrose and acacia or tragacanth; pastilles comprising the activeingredient in an inert basis such as gelatin and glycerin, or sucroseand acacia; and mouthwashes comprising the active ingredient in asuitable liquid carrier.

Pharmaceutical compositions for topical administration according to thepresent invention are optionally formulated as ointments, creams,suspensions, lotions, powders, solutions, pastes, gels, sprays, aerosolsor oils. In alternatively embodiments, topical formulations comprisepatches or dressings such as a bandage or adhesive plasters impregnatedwith active ingredient(s), and optionally one or more excipients ordiluents. In preferred embodiments, the topical formulations include acompound(s) that enhances absorption or penetration of the activeagent(s) through the skin or other affected areas. Examples of suchdermal penetration enhancers include dimethylsulfoxide (DMSO) andrelated analogues.

If desired, the aqueous phase of a cream base includes, for example, atleast about 30% w/w of a polyhydric alcohol, i.e., an alcohol having twoor more hydroxyl groups such as propylene glycol, butane-1,3-diol,mannitol, sorbitol, glycerol and polyethylene glycol and mixturesthereof In some embodiments, oily phase emulsions of this invention areconstituted from known ingredients in an known manner. This phasetypically comprises an lone emulsifier (otherwise known as an emulgent),it is also desirable in some embodiments for this phase to furthercomprises a mixture of at least one emulsifier with a fat or an oil orwith both a fat and an oil.

Preferably, a hydrophilic emulsifier is included together with alipophilic emulsifier so as to act as a stabilizer. It some embodimentsit is also preferable to include both an oil and a fat. Together, theemulsifier(s) with or without stabilizer(s) make up the so-calledemulsifying wax, and the wax together with the oil and/or fat make upthe so-called emulsifying ointment base which forms the oily dispersedphase of the cream formulations.

Emulgents and emulsion stabilizers suitable for use in the formulationof the present invention include Tween 60, Span 80, cetostearyl alcohol,myristyl alcohol, glyceryl monostearate and sodium lauryl sulfate.

The choice of suitable oils or fats for the formulation is based onachieving the desired properties (e.g., cosmetic properties), since thesolubility of the active compound/agent in most oils likely to be usedin pharmaceutical emulsion formulations is very low. Thus creams shouldpreferably be a non-greasy, non-staining and washable products withsuitable consistency to avoid leakage from tubes or other containers.Straight or branched chain, mono- or dibasic alkyl esters such asdi-isoadipate, isocetyl stearate, propylene glycol diester of coconutfatty acids, isopropyl myristate, decyl oleate, isopropyl palmitate,butyl stearate, 2-ethylhexyl palmitate or a blend of branched chainesters known as Crodamol CAP may be used, the last three being preferredesters. These may be used alone or in combination depending on theproperties required. Alternatively, high melting point lipids such aswhite soft paraffin and/or liquid paraffin or other mineral oils can beused.

Formulations suitable for topical administration to the eye also includeeye drops wherein the active ingredient is dissolved or suspended in asuitable carrier, especially an aqueous solvent for the agent.

Formulations for rectal administration may be presented as a suppositorywith suitable base comprising, for example, cocoa butter or asalicylate.

Formulations suitable for vaginal administration may be presented aspessaries, creams, gels, pastes, foams or spray formulations containingin addition to the agent, such carriers as are known in the art to beappropriate.

Formulations suitable for nasal administration, wherein the carrier is asolid, include coarse powders having a particle size, for example, inthe range of about 20 to about 500 microns which are administered in themanner in which snuff is taken, i.e., by rapid inhalation (e.g., forced)through the nasal passage from a container of the powder held close upto the nose. Other suitable formulations wherein the carrier is a liquidfor administration include, but are not limited to, nasal sprays, drops,or aerosols by nebulizer, an include aqueous or oily solutions of theagents.

Formulations suitable for parenteral administration include aqueous andnon-aqueous isotonic sterile injection solutions which may containantioxidants, buffers, bacteriostats and solutes which render theformulation isotonic with the blood of the intended recipient; andaqueous and non-aqueous sterile suspensions which may include suspendingagents and thickening agents, and liposomes or other microparticulatesystems which are designed to target the compound to blood components orone or more organs. In some embodiments, the formulations arepresented/formulated in unit-dose or multi-dose sealed containers, forexample, ampoules and vials, and may be stored in a freeze-dried(lyophilized) condition requiring only the addition of the sterileliquid carrier, for example water for injections, immediately prior touse. Extemporaneous injection solutions and suspensions may be preparedfrom sterile powders, granules and tablets of the kind previouslydescribed.

Preferred unit dosage formulations are those containing a daily dose orunit, daily subdose, as herein above-recited, or an appropriate fractionthereof, of an agent.

It should be understood that in addition to the ingredients particularlymentioned above, the formulations of this invention may include otheragents conventional in the art having regard to the type of formulationin question, for example, those suitable for oral administration mayinclude such further agents as sweeteners, thickeners and flavoringagents. It also is intended that the agents, compositions and methods ofthis invention be combined with other suitable compositions andtherapies. Still other formulations optionally include food additives(suitable sweeteners, flavorings, colorings, etc.), phytonutrients(e.g., flax seed oil), minerals (e.g., Ca, Fe, K, etc.), vitamins, andother acceptable compositions (e.g., conjugated linoelic acid),extenders, and stabilizers, etc.

C. Exemplary Administration Routes and Dosing Considerations

Various delivery systems are known and can be used to administer atherapeutic agents (e.g., benzodiazepine crystal forms and formulationsand benzodiazepine related crystal forms and formulations) of thepresent invention, e.g., encapsulation in liposomes, microparticles,microcapsules, receptor-mediated endocytosis, and the like. Methods ofdelivery include, but are not limited to, intra-arterial,intra-muscular, intravenous, intranasal, and oral routes. In specificembodiments, it may be desirable to administer the pharmaceuticalcompositions of the invention locally to the area in need of treatment;this may be achieved by, for example, and not by way of limitation,local infusion during surgery, injection, or by means of a catheter.

The agents identified herein as effective for their intended purpose canbe administered to subjects or individuals susceptible to or at risk ofdeveloping pathological growth of target cells and condition correlatedwith this. When the agent is administered to a subject such as a mouse,a rat or a human patient, the agent can be added to a pharmaceuticallyacceptable carrier and systemically or topically administered to thesubject. To determine patients that can be beneficially treated, atissue sample is removed from the patient and the cells are assayed forsensitivity to the agent.

Therapeutic amounts are empirically determined and vary with thepathology being treated, the subject being treated and the efficacy andtoxicity of the agent. When delivered to an animal, the method is usefulto further confirm efficacy of the agent. One example of an animal modelis MLR/MpJ-lpr/lpr (“MLR-lpr”) (available from Jackson Laboratories, BalHarbor, Me.). MLR-lpr mice develop systemic autoimmune disease.Alternatively, other animal models can be developed by inducing tumorgrowth, for example, by subcutaneously inoculating nude mice with about10⁵ to about 10⁹ hyperproliferative, cancer or target cells as definedherein. When the tumor is established, the compounds described hereinare administered, for example, by subcutaneous injection around thetumor. Tumor measurements to determine reduction of tumor size are madein two dimensions using venier calipers twice a week. Other animalmodels may also be employed as appropriate. Such animal models for theabove-described diseases and conditions are well-known in the art.

In some embodiments, in vivo administration is effected in one dose,continuously or intermittently throughout the course of treatment.Methods of determining the most effective means and dosage ofadministration are well known to those of skill in the art and vary withthe composition used for therapy, the purpose of the therapy, the targetcell being treated, and the subject being treated. Single or multipleadministrations are carried out with the dose level and pattern beingselected by the treating physician.

Suitable dosage formulations and methods of administering the agents arereadily determined by those of skill in the art. Preferably, thecompounds are administered at about 0.01 mg/kg to about 200 mg/kg, morepreferably at about 0.1 mg/kg to about 100 mg/kg, even more preferablyat about 0.5 mg/kg to about 50 mg/kg. When the compounds describedherein are co-administered with another agent (e.g., as sensitizingagents), the effective amount may be less than when the agent is usedalone.

The pharmaceutical compositions can be administered orally,intranasally, parenterally or by inhalation therapy, and may take theform of tablets, lozenges, granules, capsules, pills, ampoules,suppositories or aerosol form. They may also take the form ofsuspensions, solutions and emulsions of the active ingredient in aqueousor nonaqueous diluents, syrups, granulates or powders. In addition to anagent of the present invention, the pharmaceutical compositions can alsocontain other pharmaceutically active compounds or a plurality ofcompounds of the invention.

More particularly, an agent of the present invention also referred toherein as the active ingredient, may be administered for therapy by anysuitable route including, but not limited to, oral, rectal, nasal,topical (including, but not limited to, transdermal, aerosol, buccal andsublingual), vaginal, parental (including, but not limited to,subcutaneous, intramuscular, intravenous and intradermal) and pulmonary.It is also appreciated that the preferred route varies with thecondition and age of the recipient, and the disease being treated.

Ideally, the agent should be administered to achieve peak concentrationsof the active compound at sites of disease. This may be achieved, forexample, by the intravenous injection of the agent, optionally insaline, or orally administered, for example, as a tablet, capsule orsyrup containing the active ingredient.

Desirable blood levels of the agent may be maintained by a continuousinfusion to provide a therapeutic amount of the active ingredient withindisease tissue. The use of operative combinations is contemplated toprovide therapeutic combinations requiring a lower total dosage of eachcomponent antiviral agent than may be required when each individualtherapeutic compound or drug is used alone, thereby reducing adverseeffects.

D. Exemplary Co-Administration Routes and Dosing Considerations

The present invention also includes methods involving co-administrationof the compounds described herein with one or more additional activeagents. Indeed, it is a further aspect of this invention to providemethods for enhancing prior art therapies and/or pharmaceuticalcompositions by co-administering a compound of this invention. Inco-administration procedures, the agents may be administeredconcurrently or sequentially. In one embodiment, the compounds describedherein are administered prior to the other active agent(s). Thepharmaceutical formulations and modes of administration may be any ofthose described above. In addition, the two or more co-administeredchemical agents, biological agents or radiation may each be administeredusing different modes or different formulations.

The agent or agents to be co-administered depends on the type ofcondition being treated. For example, when the condition being treatedis cancer, the additional agent can be a chemotherapeutic agent orradiation. When the condition being treated is an autoimmune disorder,the additional agent can be an immunosuppressant or an anti-inflammatoryagent. When the condition being treated is chronic inflammation, theadditional agent can be an anti-inflammatory agent. The additionalagents to be co-administered, such as anticancer, immunosuppressant,anti-inflammatory, and can be any of the well-known agents in the art,including, but not limited to, those that are currently in clinical use.The determination of appropriate type and dosage of radiation treatmentis also within the skill in the art or can be determined with relativeease.

Treatment of the various conditions associated with abnormal apoptosisis generally limited by the following two major factors: (1) thedevelopment of drug resistance and (2) the toxicity of known therapeuticagents. In certain cancers, for example, resistance to chemicals andradiation therapy has been shown to be associated with inhibition ofapoptosis. Some therapeutic agents have deleterious side effects,including non-specific lymphotoxicity, renal and bone marrow toxicity.

The methods described herein address both these problems. Drugresistance, where increasing dosages are required to achieve therapeuticbenefit, is overcome by co-administering the compounds described hereinwith the known agent. The compounds described herein appear to sensitizetarget cells to known agents (and vice versa) and, accordingly, less ofthese agents are needed to achieve a therapeutic benefit.

The sensitizing function of the claimed compounds also addresses theproblems associated with toxic effects of known therapeutics. Ininstances where the known agent is toxic, it is desirable to limit thedosages administered in all cases, and particularly in those cases weredrug resistance has increased the requisite dosage. When the claimedcompounds are co-administered with the known agent, they reduce thedosage required which, in turn, reduces the deleterious effects.Further, because the claimed compounds are themselves both effective andnon-toxic in large doses, co-administration of proportionally more ofthese compounds than known toxic therapeutics will achieve the desiredeffects while minimizing toxic effects.

V. Drug Screens

In preferred embodiments of the present invention, the compounds of thepresent invention, and other potentially useful compounds, are screenedfor their biological activity (e.g., ability to initiate cell deathalone or in combination with other compounds). In preferred embodimentsof the present invention, the compounds of the present invention, andother potentially useful compounds, are screened for their bindingaffinity to the oligomycin sensitivity conferring protein (OSCP) portionof the mitochondrial ATP synthase complex. In particularly preferredembodiments, compounds are selected for use in the methods of thepresent invention by measuring their biding affinity to recombinant OSCPprotein. A number of suitable screens for measuring the binding affinityof drugs and other small molecules to receptors are known in the art. Insome embodiments, binding affinity screens are conducted in in vitrosystems. In other embodiments, these screens are conducted in in vivo orex vivo systems. While in some embodiments quantifying the intracellularlevel of ATP following administration of the compounds of the presentinvention provides an indication of the efficacy of the methods,preferred embodiments of the present invention do not requireintracellular ATP or pH level quantification.

Additional embodiments are directed to measuring levels (e.g.,intracellular) of superoxide in cells and/or tissues to measure theeffectiveness of particular contemplated methods and compounds of thepresent invention. In this regard, those skilled in the art willappreciate and be able to provide a number of assays and methods usefulfor measuring superoxide levels in cells and/or tissues.

In some embodiments, structure-based virtual screening methodologies arecontemplated for predicting the binding affinity of compounds of thepresent invention with OSCP.

In some embodiments, compounds are screened in cell culture or in vivo(e.g., non-human or human mammals) for their ability to modulatemitochondrial ATP synthase activity. Any suitable assay may be utilized,including, but not limited to, cell proliferation assays (Commerciallyavailable from, e.g., Promega, Madison, Wis. and Stratagene, La Jolla,Calif.) and cell based dimerization assays. (See e.g., Fuh et al.,Science, 256:1677 [1992]; Colosi et al., J. Biol. Chem., 268:12617[1993]). Additional assay formats that find use with the presentinvention include, but are not limited to, assays for measuring cellularATP levels, and cellular superoxide levels.

Any suitable assay that allows for a measurement of the rate of bindingor the affinity of a benzodiazepine or other compound to the OSCP may beutilized. Examples include, but are not limited to, competition bindingusing Bz-423, surface plasma resonace (SPR) andradio-immunopreciptiation assays (Lowman et al, J. Biol. Chem. 266:10982[1991]). Surface Plasmon Resonance techniques involve a surface coatedwith a thin film of a conductive metal, such as gold, silver, chrome oraluminum, in which electromagnetic waves, called Surface Plasmons, canbe induced by a beam of light incident on the metal glass interface at aspecific angle called the Surface Plasmon Resonance angle. Modulation ofthe refractive index of the interfacial region between the solution andthe metal surface following binding of the captured macromoleculescauses a change in the SPR angle which can either be measured directlyor which causes the amount of light reflected from the underside of themetal surface to change. Such changes can be directly related to themass and other optical properties of the molecules binding to the SPRdevice surface. Several biosensor systems based on such principles havebeen disclosed (See e.g., WO 90/05305). There are also severalcommercially available SPR biosensors (e.g., BiaCore, Uppsala, Sweden).

In some embodiments, compounds are screened in cell culture or in vivo(e.g., non-human or human mammals) for their ability to modulatemitochondrial ATP synthase activity. Any suitable assay may be utilized,including, but not limited to, cell proliferation assays (Commerciallyavailable from, e.g., Promega, Madison, Wis. and Stratagene, La Jolla,Calif.) and cell based dimerization assays. (See e.g., Fuh et al.,Science, 256:1677 [1992]; Colosi et al., J. Biol. Chem., 268:12617[1993]). Additional assay formats that find use with the presentinvention include, but are not limited to, assays for measuring cellularATP levels, and cellular superoxide levels.

The present invention also provides methods of modifying andderivatizing the compositions of the present invention to increasedesirable properties (e.g., binding affinity, activity, and the like),or to minimize undesirable properties (e.g., nonspecific reactivity,toxicity, and the like). The principles of chemical derivatization arewell understood. In some embodiments, iterative design and chemicalsynthesis approaches are used to produce a library of derivatized childcompounds from a parent compound. In other embodiments, rational designmethods are used to predict and model in silico ligand-receptorinteractions prior to confirming results by routine experimentation.

VI. Therapeutic Application

A. General Therapeutic Application

In particularly preferred embodiments, the compositions of the presentinvention provide therapeutic benefits to patients suffering from anyone or more of a number of conditions (e.g., diseases characterized bydysregulation of necrosis and/or apoptosis processes in a cell ortissue, disease characterized by aberrant cell growth and/orhyperproliferation, etc.) by modulating (e.g., inhibiting or promoting)the activity of the mitochondrial ATP synthase (as referred to asmitochondrial F₀F₁ ATPase) complexes in affected cells or tissues (e.g.,myocardial infarction, ventricular hypertrophy, coronary artery disease,non-Q wave MI, congestive heart failure, cardiac arrhythmias, unstableangina, chronic stable angina, Prinzmetal's angina, high blood pressure,intermittent claudication, peripheral occlusive arterial disease,thrombotic or thromboembolic symptoms of thromboembolic stroke, venousthrombosis, arterial thrombosis, cerebral thrombosis, pulmonaryembolism, cerebral embolism, thrombophilia, disseminated intravascularcoagulation, restenosis, atrial fibrillation, ventricular enlargement,atherosclerotic vascular disease, atherosclerotic plaque rupture,atherosclerotic plaque formation, transplant atherosclerosis, vascularremodeling atherosclerosis, cancer, surgery, inflammation, systematicinfection, artificial surfaces, interventional cardiology, immobility,medication, pregnancy and fetal loss, and diabetic complicationscomprising retinopathy, nephropathy and neuropathy). In furtherpreferred embodiments, the compositions of the present invention areused to treat autoimmune/chronic inflammatory conditions (e.g.,psoriasis). In even further embodiments, the compositions of the presentinvention are used in conjunction with stenosis therapy to treatcompromised (e.g., occluded) vessels. Indeed, any application wherebenzodiazepines find use is contemplated by the present invention.

In particularly preferred embodiments, the compositions of the presentinvention inhibit the activity of mitochondrial ATP synthase complex bybinding to a specific subunit or subunits of this multi-subunit proteincomplex. While the present invention is not limited to any particularmechanism, nor to any understanding of the action of the agents beingadministered, in some embodiments, it is contemplated that thecompositions of the present invention bind to the oligomycin sensitivityconferring protein (OSCP) portion of the mitochondrial ATP synthasecomplex, to the OSCP/F1 junction, or to the F1 subunit. Likewise, it isfurther contemplated that when the compositions of the present inventionbind to the OSCP the initial affect is overall inhibition of themitochondrial ATP synthase complex, and that the downstream consequenceof binding is a change in ATP or pH level and the production of reactiveoxygen species (e.g., O₂—). In still other preferred embodiments, whilethe present invention is not limited to any particular mechanism, nor toany understanding of the action of the agents being administered, it iscontemplated that the generation of free radicals ultimately results incell killing. In yet other embodiments, while the present invention isnot limited to any particular mechanism, nor to any understanding of theaction of the agents being administered, it is contemplated that theinhibiting mitochondrial ATP synthase complex using the compositions andmethods of the present invention provides therapeutically usefulinhibition of cell proliferation.

Accordingly, preferred methods embodied in the present invention,provide therapeutic benefits to patients by providing compounds of thepresent invention that modulate (e.g., inhibiting or promoting) theactivity of the mitochondrial ATP synthase complexes in affected cellsor tissues via binding to the oligomycin sensitivity conferring protein(OSCP) portion of the mitochondrial ATP synthase complex. Importantly,by itself the OSCP, the OSCP/F1 junction, or the F1 subunit has nobiological activity.

Thus, in one broad sense, preferred embodiments of the present inventionare directed to the discovery that many diseases characterized bydysregulation of necrosis and/or apoptosis processes in a cell ortissue, or diseases characterized by aberrant cell growth and/orhyperproliferation, etc., can be treated by modulating the activity ofthe mitochondrial ATP synthase complex including, but not limited to, bybinding to the oligomycin sensitivity conferring protein (OSCP)/F1components thereof. The present invention is not intended to be limited,however, to the practice of the compositions and methods explicitlydescribed herein. Indeed, those skilled in the art will appreciate thata number of additional compounds not specifically recited herein (e.g.,non-benzodiazepine derivatives) are suitable for use in the methodsdisclosed herein of modulating the activity of mitochondrial ATPsynthase.

The present invention thus specifically contemplates that any number ofsuitable compounds presently known in the art, or developed later, canoptionally find use in the methods of the present invention. Forexample, compounds including, but not limited to, oligomycin, ossamycin,cytovaricin, apoptolidin, bafilomyxcin, resveratrol, piceatannol, anddicyclohexylcarbodiimide (DCCD), and the like, find use in the methodsof the present invention. The present invention is not intended,however, to be limited to the methods or compounds specified above. Inone embodiment, that compounds potentially useful in the methods of thepresent invention may be selected from those suitable as described inthe scientific literature. (See e.g., K. B. Wallace and A. A. Starkov,Annu. Rev. Pharmacol. Toxicol., 40:353-388 [2000]; A. R. Solomon et al.,Proc. Nat. Acad. Sci. U.S.A., 97(26): 14766-14771 [2000]).

In some embodiments, compounds potentially useful in methods of thepresent invention are screened against the National Cancer Institute's(NCI-60) cancer cell lines for efficacy. (See e.g., A. Monks et al., J.Natl. Cancer Inst., 83:757-766 [1991]; and K. D. Paull et al., J. Natl.Cancer Inst., 81:1088-1092 [1989]). Additional screens suitable screens(e.g., autoimmunity disease models, etc.) are within the skill in theart.

In one aspect, derivatives (e.g., pharmaceutically acceptable salts,analogs, stereoisomers, and the like) of the exemplary compounds orother suitable compounds are also contemplated as being useful in themethods of the present invention.

In other preferred embodiments, the compositions of the presentinvention are used in conjunction with stenosis therapy to treatcompromised (e.g., occluded) vessels. In further embodiments, thecompositions of the present invention are used in conjunction withstenosis therapy to treat compromised cardiac vessels.

Vessel stenosis is a condition that develops when a vessel (e.g., aorticvalve) becomes narrowed. For example, aortic valve stenosis is a heartcondition that develops when the valve between the lower left chamber(left ventricle) of the heart and the major blood vessel called theaorta becomes narrowed. This narrowing (e.g., stenosis) creates toosmall a space for the blood to flow to the body. Normally the leftventricle pumps oxygen-rich blood to the body through the aorta, whichbranches into a system of arteries throughout the body. When the heartpumps, the 3 flaps, or leaflets, of the aortic valve open one way toallow blood to flow from the ventricle into the aorta. Betweenheartbeats, the flaps close to form a tight seal so that blood does notleak backward through the valve. If the aortic valve is damaged, it maybecome narrowed (stenosed) and blood flow may be reduced to organs inthe body, including the heart itself. The long-term outlook for peoplewith aortic valve stenosis is poor once symptoms develop. People withuntreated aortic valve stenosis who develop symptoms of heart failureusually have a life expectancy of 3 years or less.

Several types of treatment exist for treating compromised valves (e.g.,balloon dilation, ablation, atherectomy or laser treatment). One type oftreatment for compromised cardiac valves is angioplasty. Angioplastyinvolves inserting a balloon-tipped tube, or catheter, into a narrow orblocked artery in an attempt to open it. By inflating and deflating theballoon several times, physicians usually are able to widen the artery.

A common limitation of angioplasty or valve expansion procedures isrestenosis. Restenosis is the reclosure of a peripheral or coronaryartery following trauma to that artery caused by efforts to open astenosed portion of the artery, such as, for example, by balloondilation, ablation, atherectomy or laser treatment of the artery. Forthese angioplasty procedures, restenosis occurs at a rate of about20-50% depending on the definition, vessel location, lesion length and anumber of other morphological and clinical variables. Restenosis isbelieved to be a natural healing reaction to the injury of the arterialwall that is caused by angioplasty procedures. The healing reactionbegins with the thrombotic mechanism at the site of the injury. Thefinal result of the complex steps of the healing process can be intimalhyperplasia, the uncontrolled migration and proliferation of medialsmooth muscle cells, combined with their extracellular matrixproduction, until the artery is again stenosed or occluded.

In an attempt to prevent restenosis, metallic intravascular stents havebeen permanently implanted in coronary or peripheral vessels. The stentis typically inserted by catheter into a vascular lumen told expandedinto contact with the diseased portion of the arterial wall, therebyproviding mechanical support for the lumen. However, it has been foundthat restenosis can still occur with such stents in place. Also, thestent itself can cause undesirable local thrombosis. To address theproblem of thrombosis, persons receiving stents also receive extensivesystemic treatment with anticoagulant and antiplatelet drugs.

To address the restenosis problem, it has been proposed to providestents which are seeded with endothelial cells (Dichek, D. A. et alSeeding of Intravascular Stents With Genetically Engineered EndothelialCells; Circulation 1989; 80: 1347-1353). In that experiment, sheependothelial cells that had undergone retrovirus-mediated gene transferfor either bacterial beta-galactosidase or human tissue-type plasminogenactivator were seeded onto stainless steel stents and grown until thestents were covered. The cells were therefore able to be delivered tothe vascular wall where they could provide therapeutic proteins. Othermethods of providing therapeutic substances to the vascular wall bymeans of stents have also been proposed such as in international patentapplication WO 91/12779 “Intraluminal Drug Eluting Prosthesis” andinternational patent application WO 90/13332 “Stent With Sustained DrugDelivery”. In those applications, it is suggested that antiplateletagents, anticoagulant agents, antimicrobial agents, anti-inflammatoryagents, antimetabolic agents and other drugs could be supplied in stentsto reduce the incidence of restenosis. Further, other vasoreactiveagents such as nitric oxide releasing agents could also be used.

An additional cause of restenosis is the over-proliferation of treatedtissue. In preferred embodiments, the anti-proliferative properties ofthe present invention inhibit restenosis. Drug-eluting stents are wellknown in the art (see, e.g., U.S. Pat. No. 5,697,967;U.S. Pat. No.5,599,352; and U.S. Pat. No. 5,591,227; each of which are hereinincorporated by reference). In preferred embodiments, the compositionsof the present invention are eluted from drug-eluting stents in thetreatment of compromised (e.g., occluded) vessels. In furtherembodiments, the compositions of the present invention are eluted fromdrug-eluting stents in the treatment of compromised cardiac vessels.

Those skilled in the art of preparing pharmaceutical compounds andformulations will appreciate that when selecting optional compounds foruse in the methods disclosed herein, that suitability considerationsinclude, but are not limited to, the toxicity, safety, efficacy,availability, and cost of the particular compounds.

In preferred embodiments, pharmaceutical compositions comprise compoundsof the invention and, for example, therapeutic agents (e.g.,antiatherosclerotic agents, anticoagulants, antithrombotic agents,antihypertensive agents, and antidiabetic agents). Antihypertensiveagents include, but are not limited to, ACE inhibitors, AT-1 receptorantagonists, ET receptor antagonists, dual ET/AII receptor antagonists,and vasopepsidase inhibitors, or an antiplatelet agent selected fromGPIIb/IIIa blockers, P2Y₁ and P2Y₁₂ antagonists, thromboxane receptorantagonists, and aspirin.

In preferred embodiments, the compounds of the present invention areuseful in treating a mitochondrial F₁F₀ ATP hydrolase associateddisorder (e.g., myocardial infarction, ventricular hypertrophy, coronaryartery disease, non-Q wave MI, congestive heart failure, cardiacarrhythmias, unstable angina, chronic stable angina, Prinzmetal'sangina, high blood pressure, intermittent claudication, peripheralocclusive arterial disease, thrombotic or thromboembolic symptoms ofthromboembolic stroke, venous thrombosis, arterial thrombosis, cerebralthrombosis, pulmonary embolism, cerebral embolism, thrombophilia,disseminated intravascular coagulation, restenosis, atrial fibrillation,ventricular enlargement, atherosclerotic vascular disease,atherosclerotic plaque rupture, atherosclerotic plaque formation,transplant atherosclerosis, vascular remodeling atherosclerosis, cancer,surgery, inflammation, systematic infection, artificial surfaces,interventional cardiology, immobility, medication, pregnancy and fetalloss, and diabetic complications comprising retinopathy, nephropathy andneuropathy) in a patient.

B. Autoimmune Disorder and Chronic Inflammatory Disorder TherapeuticApplication

Autoimmune disorders and chronic inflammatory disorders often resultfrom dysfunctional cellular proliferation regulation and/or cellularapoptosis regulation. Mitochondria perform a key role in the control andexecution of cellular apoptosis. The mitochondrial permeabilitytransition pore (MPTP) is a pore that spans the inner and outermitochondrial membranes and functions in the regulation of proapoptoticparticles. Transient MPTP opening results in the release of cytochrome cand the apoptosis inducing factor from the mitochondrial intermembranespace, resulting in cellular apoptosis.

The oligomycin sensitivity conferring protein (OSCP) is a subunit of theF₀F₁ mitochondrial ATP synthase/ATPase and functions in the coupling ofa proton gradient across the F₀ sector of the enzyme in themitochondrial membrane. In preferred embodiments, compounds of thepresent invention binds the OSCP, the OSCP/F1 junction, or the F1subunit increases superoxide and cytochrome c levels, increases cellularapoptosis, and inhibits cellular proliferation. The adenine nucleotidetranslocator (ANT) is a 30 kDa protein that spans the innermitochondrial membrane and is central to the mitochondrial permeabilitytransition pore (MPTP). Thiol oxidizing or alkylating agents arepowerful activators of the MPTP that act by modifying one or more ofthree unpaired cysteines in the matrix side of the ANT.4-(N-(S-glutathionylacetyl)amino)phenylarsenoxide,

inhibits the ANT.

The compounds and methods of the present invention are useful in thetreatment of autoimmune disorders and chronic inflammatory disorders. Insuch embodiments, the present invention provides a subject sufferingfrom an autoimmune disorder and/or a chronic inflammatory disorder, anda composition comprising, for example,

Additionally, in preferred embodiments, the compositions may compriseany of the compounds described in the present invention, and any of thecompounds described in U.S. Provisional Patent Nos. 60/131,761,60/165,511, 60/191,855, 60/312,560, 60/313,689, 60/396,670, 60/565,788,60/607,599, 60/641,040, and U.S. patent application Ser. Nos.11/324,419, 11/176,719, 11/110,228, 10/935,333, 10/886,450, 10/795,535,10/634,114, 10/427,211, 10/427,212, 10/217,878, 09/767,283, 09/700,101,and related applications; each herein incorporated by reference in theirentireties.

C. Treatment of Epidermal Hyperplasia

Epidermal hyperplasia (e.g., excessive keratinocyte proliferation)leading to a significant thickening of the epidermis in association withshedding of the thickened epidermis, is a feature of diseases such aspsoriasis (see, e.g., Krueger G C, et al., (1984) J. Am. Acad. Dermatol.11: 937-947; Fry L. (1988), Brit. J. Dermatol. 119:445-461; each hereinincorporated by reference in their entireties) and also occurs underphysiological conditions (e.g., during wound-healing).

Topical treatment of the skin with all-trans retinoic acid (RA) or itsprecursor, all-trans retinol (ROL) also results in epidermal hyperplasia(see, e.g., Varani J, et al., (2001) J. Invest. Dermatol, 117:1335-1341;herein incorporated by reference in its entirety). While the underlyingetiologies are different, all of these hyperplasias have in common theactivation of the epidermal growth factor (EGF) receptor in theproliferating keratinocytes (see, e.g., Varani J, et al., (2001) J.Invest. Dermatol 117:1335-1341; Baker B S, et al., (1992) Brit. J.Dermatol. 126:105-110; Gottlieb A B, et al., (1988) J. Exp. Med.167:670-675; Elder J T, et al., (1989) Science 243:811-814; Piepkorn M,et al., (1998) J Invest Dermatol 111:715-721; Piepkorn M, et al., (2003)Arch Dermatol Res 27:27; Cook P W, et al., (1992) Cancer Res52:3224-3227; each herein incorporated by reference in theirentireties). Normal epidermal growth does not appear to be as dependenton EGF receptor function as hyperplastic growth (see, e.g., Varani J, etal., (2001) J. Invest. Dermatol 117:1335-1341; Varani J, et al., (1998)Pathobiology 66:253-259; each herein incorporated by reference in theirentireties). Likewise, function of the dermis in intact skin does notdepend on EGF receptor function (see, e.g., Varani J, et al., (2001) J.Invest. Dermatol 117:1335-1341; herein incorporated by reference in itsentirety).

The central role of the EGF receptor in regulating hyperplasticepithelial growth makes the EGF receptor tyrosine kinase a target forantiproliferative agents. Likewise, the series of signaling moleculesengaged downstream of this receptor are additional points at whichkeratinocyte growth can be interrupted. The mitogen activated proteinkinase (MAPK) cascade is activated by the EGF receptor (see, e.g.,Marques, S. A., et al., (2002) J Pharmacol Exp Ther 300, 1026-1035;herein incorporated by reference in its entirety). In hyperproliferativeepidermis, but not in normal epidermis, extracellular signal-regulatedkinases 1/2 (Erk 1/2) are activated in basal and suprabasalkeratinocytes and contribute to epidermal hyperproliferation (see, e.g.,Haase, I., et al., (2001) J Clin Invest 108, 527-536; Takahashi, H., etal., (2002) J Dermatol Sci 30, 94-99; each herein incorporated byreference in their entireties). In culture models, keratinocyte growthregulation through the EGF receptor results in increased MAPK activity.In keratinocytes, growth factor-stimulated MAPK activity is alsodependent on integrin engagement and extracellular matrix molecules thatbind integrins are capable of independently activating MAPKs andincreasing keratinocyte proliferation (see, e.g., Haase, I., et al.,(2001) J Clin Invest 108, 527-536; herein incorporated by reference inits entirety). The proliferation of other skin cells, includingfibroblasts, is less dependent on Erk 1/2 activity, making Erkinhibition a potentially useful characteristic to evaluate leadcompounds for potential utility against epidermal hyperplasia.

In preferred embodiments, compounds of the present invention are usedfor treating epidermal hyperplasias.

In preferred embodiments, compounds of the present invention are used intreating psoriasis. Psoriasis is common and chronic epidermalhyperplasia. Plaque psoriasis is the most common type of psoriasis andis characterized by red skin covered with silvery scales andinflammation. Patches of circular to oval shaped red plaques that itchor burn are typical of plaque psoriasis. The patches are usually foundon the arms, legs, trunk, or scalp but may be found on any part of theskin. The most typical areas are the knees and elbows. Psoriasis is notcontagious and can be inherited. Environmental factors, such as smoking,sun exposure, alcoholism, and HIV infection, may affect how often thepsoriasis occurs and how long the flares up last.

Treatment of psoriasis includes topical steroids, coal tar, keratolyticagents, vitamin D-3 analogs, and topical retinoids. Topical steroids areagents used to reduce plaque formation. Topical steroid agents haveanti-inflammatory effects and may cause profound and varied metabolicactivities. In addition, topical steroid agents modify the body's immuneresponse to diverse stimuli. Examples of topical steroids include, butare not limited to, triamcinolone acetonide (Artistocort, Kenalog) 0.1%cream, and betamethasone diproprionate (Diprolene, Diprosone) 0.05%cream. Coal tar is an inexpensive treatment available over the counterin shampoos or lotions for use in widespread areas of involvement. Coaltar is particularly useful in hair-bearing areas. An example of coal taris coal tar 2-10% (DHS Tar, Doctar, Theraplex T)—antipruitic.Keratolytic agents are used to remove scale, smooth the skin, and totreat hyperkeratosis. An example of a keratolytic agent is anthralin0.1-1% (Drithocreme, Anthra-Derm). Vitamin D-3 analogs are used inpatients with lesions resistant to older therapy or with lesions on theface or exposed areas where thinning of the skin would pose cosmeticproblems. An example of a vitamin D-3 analog is calcipotriene (Dovonex).Topical retinoids are agents that decrease the cohesiveness offollicular epithelial cells and stimulate mitotic activity, resulting inan increase in turnover of follicular epithelial cells. Examples oftopical retinoids include, but are not limited to, tretinoin (Retin-A,Avita), and tazarotene (Tazorac).

Approximately 1-2% of people in the United States, or about 5.5 million,have plaque psoriasis. Up to 30% of people with plaque psoriasis alsohave psoriatic arthritis. Individuals with psoriatic arthritis haveinflammation in their joints and may have other arthritis symptoms.Sometimes plaque psoriasis can evolve into more severe disease, such aspustular psoriasis or erythrodermic psoriasis. In pustular psoriasis,the red areas on the skin contain blisters with pus. In erythrodermicpsoriasis, a wide area of red and scaling skin is typical, and it may beitchy and painful. The present invention is useful in treatingadditional types of psoriasis, including but not limited to, guttatepsoriasis, nail psoriasis, inverse psoriasis, and scalp psoriasis.

In some embodiments, the compounds of the present invention are usefulin treating pigmentation disorders (e.g., albinism, melasma, andvitiligo). The present invention is not limited to a particularmechanism for treating pigment disorders. In preferred embodiments,pigment disorders are treated through targeting of the F₁F_(o)-ATPase bythe compounds of the present invention. In further embodiments, pigmentdisorders are treated through the rerouting of tyrosinase by thecompounds of the present invention. In further embodiments, pigmentdisorders are treated through targeting of prohibitin by the compoundsof the present invention.

VII. ATPase Inhibitors and Methods for Identifying TherapeuticInhibitors

The present invention provides compounds that target the F₁F_(o)-ATPase.In addition, the present invention provides compounds that target theF₁F_(o)-ATPase as a treatment for autoimmune disorders, and inparticular, compounds with low toxicity. The present invention furtherprovides methods of identifying compounds that target theF₁F_(o)-ATPase. Additionally, the present invention provides therapeuticapplications for compounds targeting the F₁F_(o)-ATPase.

A majority of ATP within eukaryotic cells is synthesized by themitochondrial F₁F_(o)-ATPase (see, e.g., C. T. Gregory et al., J.Immunol., 139:313-318 [1987]; J. P. Portanova et al., Mol. Immunol.,32:117-135 [1987]; M. J. Shlomchik et al., Nat. Rev. Immunol., 1:147-153[2001]; each herein incorporated by reference in their entireties).Although the F₁F_(o)-ATPase synthesizes and hydrolyzes ATP, duringnormal physiologic conditions, the F₁F_(o)-ATPase only synthesizes ATP(see, e.g., Nagyvary J, et al., Biochem. Educ. 1999; 27:193-99; hereinincorporated by reference in its entirety). The mitochondrialF₁F_(o)-ATPase is composed of three major domains: F_(o), F₁ and theperipheral stator. F₁ is the portion of the enzyme that contains thecatalytic sites and it is located in the matrix (see, e.g., Boyer, P D,Annu Rev Biochem.1997; 66:717-49; herein incorporated by reference inits entirety). This domain is highly conserved and has the subunitcomposition α₃β₃γδε. The landmark X-ray structure of bovine F₁ revealedthat α₃β₃ forms a hexagonal cylinder with the γ subunit in the center ofthe cylinder. F_(o) is located within the inner mitochondrial membraneand contains a proton channel. Translocation of protons from theinner-membrane space into the matrix provides the energy to drive ATPsynthesis. The peripheral stator is composed of several proteins thatphysically and functionally link F_(o) with F₁. The stator transmitsconformational changes from F_(o) into in the catalytic domain thatregulate ATP synthesis (see, e.g., Cross R L, Biochim Biophys Acta 2000;1458:270-75; herein incorporated by reference in its entirety).

Mitochondrial F₁F_(o)-ATPase inhibitors are invaluable tools formechanistic studies of the F₁F_(o)-ATPase (see, e.g., James A M, et al.,J Biomed Sci 2002; 9:475-87; herein incorporated by reference in itsentirety). Because F₁F_(o)-ATPase inhibitors are often cytotoxic, theyhave been explored as drugs for cancer and other hyperproliferativedisorders. Macrolides (e.g., oligomycin and apoptolidin) arenon-competitive inhibitors of the F₁F_(o)-ATPase (see, e.g., Salomon AR, et al., PNAS 2000; 97:14766-71; Salomon A R, et al., Chem Biol 2001;8:71-80; herein incorporated by reference in its entirety). Macrolidesbind to F_(o) which blocks proton flow through the channel resulting ininhibition of the F₁F_(o)-ATPase. Macrolides are potent (e.g., the IC₅₀for oligomycin=10 nM) and lead to large decreases in [ATP]. As such,macrolides have an unacceptably narrow therapeutic index and are highlytoxic (e.g., the LD₅₀ for oligomycin in rodents is two daily doses at0.5 mg/kg) (see, e.g., Kramar R, et al., Agents & Actions 1984,15:660-63; herein incorporated by reference in its entirety). Otherinhibitors of F₁F_(o)-ATPase include Bz-423, which binds to the OSCP inF₁ (as described elsewhere herein). Bz-423 has an K_(i)˜9 μM. Bz-423 isdescribed in U.S. patent application Ser. Nos. 10/634,114, 10/427,211,10/427,212, 10/217,878, 09/767,283, 09/700,101, and relatedapplications, each herein incorporated by reference in their entireties.

In cells that are actively respiring (known as state 3 respiration),inhibiting F₁F_(o)-ATPase blocks respiration and places the mitochondriain a resting state (known as state 4). In state 4, the MRC is reducedrelative to state 3, which favors reduction of O₂ to O₂ ⁻ at complex III(see, e.g., N. Zamzami et al., J. Exp. Med., 181:1661-1672 [1995];herein incorporated by reference in its entirety). For example, treatingcells with either oligomycin leads to a rise of intracellular O₂ ⁻ as aconsequence of inhibiting complex V. In the case of oligomycin,supplementing cells with ATP protects against death whereas antioxidantsdo not, indicating that cell death results from the drop in ATP (see,e.g., Zhang J G, et al., Arch Biochem Biophys 2001; 393:87-96; McConkeyDJ, et al., The ATP switch in apoptosis. In: Nieminen La, ed.Mitochondria in pathogenesis. New York: Plenum, 2001:265-77; each hereinincorporated by reference in their entireties). Bz-423-induced celldeath is blocked by antioxidants and is not affected by supplementingcells with ATP, indicating that Bz-423 engages an ROS-dependent deathresponse (see, e.g., N. B. Blatt, et al., J. Clin. Invest., 2002, 110,1123; herein incorporated by reference in its entirety). As such,F₁F_(o)-ATPase inhibitors are either toxic (e.g., oligomycin) ortherapeutic (e.g., Bz-423).

The present invention provides a method of distinguishing toxicF₁F_(o)-ATPase inhibitors from therapeutic F₁F_(o)-ATPase inhibitors.F₁F_(o)-ATPase inhibitors with therapeutic potential present a novelmode of inhibition. Specifically, F₁F_(o)-ATPase inhibitors withbeneficial properties are uncompetitive inhibitors that only bindenzyme-substrate complexes at high substrate concentration and do notalter the k_(cat)/K_(m) ratio. This knowledge forms the basis toidentify and distinguish F₁F_(o)-ATPase inhibitors with therapeuticpotential from toxic compounds.

The present invention provides compounds that target the F₁F_(o)-ATPaseas an autoimmune disorder treatment. In particular, the presentinvention provides methods of identifying compounds that target theF₁F_(o)-ATPase while not altering the k_(cat)/K_(m) ratio. Additionally,the present invention provides therapeutic applications for compoundstargeting the F₁F_(o)-ATPase.

A. ATPase Inhibiting Compounds

The present invention provides compounds that inhibit theF₁F_(o)-ATPase. In some embodiments, the compounds do not bind freeF₁F_(o)-ATPase, but rather bind to an F₁F_(o)-ATPase-substrate complex.The compounds show maximum activity at high substrate concentration andminimal activity (e.g., F₁F_(o)-ATPase inhibiting) at low substrateconcentration. In preferred embodiments, the compounds do not alter thek_(cat)/K_(m) ratio of the F₁F_(o)-ATPase. The properties of theF₁F_(o)-ATPase inhibitors of the present invention are in contrast witholigomycin, which is a F₁F_(o)-ATPase inhibitor that is acutely toxicand lethal. Oligomycin is a noncompetitive inhibitor, which binds toboth free F₁F_(o)-ATPase and F₁F_(o)-ATPase-substrate complexes andalters the k_(cat)/K_(m) ratio.

The compounds of the present invention that inhibit F₁F_(o)-ATPase whilenot altering the k_(cat)/K_(m) ratio, in some embodiments, have thestructure described elsewhere herein. However, compounds of otherstructures that are identified as therapeutic inhibitors by the methodsof the present invention are also encompassed by the present invention.

B. Identifying ATPase Inhibitors

The present invention provides methods of identifying (e.g., screening)compounds useful in treating autoimmune disorders. The present inventionis not limited to a particular type compound. In preferred embodiments,compounds of the present invention include, but are not limited to,pharmaceutical compositions, small molecules, antibodies, largemolecules, synthetic molecules, synthetic polypeptides, syntheticpolynucleotides, synthetic nucleic acids, aptamers, polypeptides,nucleic acids, and polynucleotides. The present invention is not limitedto a particular method of identifying compounds useful in treatingautoimmune disorders. In preferred embodiments, compounds useful intreating autoimmune disorders are identified as possessing an ability toinhibit an F₁F_(o)-ATPase while not altering the k_(cat)/K_(m) ratio.

C. Therapeutic Applications With F₁F_(o)-ATPase Inhibitors

The present invention provides methods for treating disorders (e.g.,neurodegenerative diseases, Alzheimers, ischemia reprofusion injury,neuromotor disorders, non-Hodgkin's lymphoma, lymphocytic leukemia,cutaneous T cell leukemia, an autoimmune disorder, cancer, solid tumors,lymphomas, leukemias, and tuberculosis). The present invention is notlimited to a particular form of treatment. In preferred embodiments,treatment includes, but is not limited to, symptom amelioration, symptomprevention, disorder prevention, and disorder amelioration. The presentinvention provides methods of treating autoimmune disorders applicablewithin in vivo, in vitro, and/or ex vivo settings.

In some embodiments, the present invention treats autoimmune disordersthrough inhibiting of target cells. The present invention is not limitedto a particular form of cell inhibition. In preferred embodiments, cellinhibition includes, but is not limited to, cell growth prevention, cellproliferation prevention, and cell death. In preferred embodiments,inhibition of a target cell is accomplished through contacting a targetcell with an F₁F_(o)-ATPase inhibitor of the present invention. Infurther embodiments, target cell inhibition is accomplished throughtargeting of the F₁F_(o)-ATPase with an F₁F_(o)-ATPase inhibitor of thepresent invention. The present invention is not limited to a particularF₁F_(o)-ATPase inhibitor. In preferred embodiments, the F₁F_(o)-ATPaseinhibitor possesses the ability to inhibit an F₁F_(o)-ATPase while notaltering the k_(cat)/K_(m) ratio.

The present invention further provides methods for selectivelyinhibiting the pathology of target cells in a subject in need oftherapy. The present invention is not limited to a particular method ofinhibition target cell pathology. In preferred embodiments, target cellpathology is inhibited through administration of an effective amount ofa compound of the invention. The present invention is not limited to aparticular compound. In preferred embodiments, the compound is anF₁F_(o)-ATPase inhibitor. In further preferred embodiments, the compoundinhibits the F₁F_(o)-ATPase while not altering the k_(cat)/K_(m) ratio.

EXAMPLES

The following examples are provided to demonstrate and furtherillustrate certain preferred embodiments of the present invention andare not to be construed as limiting the scope thereof. Examplesillustrating the various uses and applications of benzodiazepinecompounds and benzodiazepine related compounds are described, forexample, in U.S. Provisional Patent Nos. 60/131,761, 60/165,511,60/191,855, 60/312,560, 60/313,689, 60/396,670, 60/565,788, 60/607,599,60/641,040, and U.S. patent application Ser. Nos. 11/324,419,11/176,719, 11/110,228, 10/935,333, 10/886,450, 10/795,535, 10/634,114,10/427,211, 10/427,212, 10/217,878, 09/767,283, 09/700,101, and relatedapplications; each herein incorporated by reference in their entireties.

Example 1

This example describes the formation of benzodiazepine crystal forms andformulations. Bz-423 (15.8 mg) was added to a 1 mL vial and dissolved inpolyethylene glycol dimethyl ether 500 (0.01 mL) at 88° C. The vial waskept at 88° C. for 16 hours, during which time crystals formed. The vialwas then cooled to room temperature and washed with cold ethyl ether(5×2 mL) to yield 4.2 mg of anhydrous Bz-423 crystals, a 27% yield. FIG.1 shows the structural data of anhydrous Bz-423, FIG. 2 shows powderx-ray diffraction data for anhydrous Bz-423, and FIG. 3 shows Ramanspectroscopy data for anhydrous Bz-423.

Bz-423 ethanol solvate was crystallized by evaporation of ethanol atroom temperature. FIG. 4 shows the structural data of Bz-423 ethanolsolvate, FIG. 5 shows powder x-ray diffraction data for Bz-423 ethanolsolvate, and FIG. 6 shows Raman spectroscopy data for Bz-423 ethanolsolvate.

Ball milled Bz-423 succinic acid (2:1) was crystallized fromtetraethylene glycol dimethyl ether at 88° C. FIG. 7 shows Ramanspectroscopy data for ball milled Bz-423 succinic acid (2:1).

Ball milled Bz-423 citric acid (2:1) was generated through ball millinga 2:1 mixture of Bz-423 and citric acid. FIG. 8 shows Raman spectroscopyball milled Bz-423 citric acid (2:1).

Bz-423 biphenyl derivate was crystallized from methanol at roomtemperature. FIG. 9 shows the structural data of Bz-423 biphenylderivate.

BZ-423-acetic acid was crystallized by evaporation of acetic acid atroom temperature. BZ-423-CH₃CN was crystallized by evaporation of CH₃CNat room temperature. BZ-423-methanol was crystallized by evaporation ofmethanol at room temperature. BZ-423-ethyl acetate was crystallized byevaporation of ethyl acetate at room temperature. BZ-423-toluene wascrystallized by evaporation of toluene at room temperature.BZ-423-oxalic acid was crystallized from tetraethylene glycol dimethylether at 88° C. BZ-423-fumaric acid was crystallized from tetraethyleneglycol dimethyl ether at 88° C. BZ-423-octanol was crystallized from asupersaturated solution of octanol. BZ-423-heptanoic acid wascrystallized from a supersaturated solution of heptanoic acid.BZ-423-diphenyl ether was crystallized from a supersaturated solution ofdiphenyl ether. BZ-423-trichlorobenzene was crystallized from asupersaturated solution of trichlorobenzene.

Example 2

This example demonstrates that anhydrous Bz-423 is more soluble thansolvated Bz-423. Water solubility at 37° C. analyses were conducted forsolvated Bz-423, anhydrous Bz-423, Bz-423 acetic acid, and Bz-423 citricacid. Table 1 shows the solubility results for solvated Bz-423,anhydrous Bz-423, Bz-423 acetic acid, Bz-423 citric acid, and Bz-423micronized.

TABLE 1 as supplied BZ-423 ball milled 1.00 anhydrous BZ-423 ball milled1.4 BZ-423 acetic acid 1.4 BZ-423 citric acid 1.4 Bz-423 micronized 0.85FIG. 10 shows solubility (e.g., absorbance) as a function of time forsolvated Bz-423, anhydrous Bz-423, Bz-423 acetic acid, and Bz-423 citricacid.

Example 3

This example demonstrates that unsolvated Bz-423 is capable ofinhibiting ATP hydrolysis, not inhibiting cell synthesis, not affectingcell viability, and its activity is related to binding of the OSCP,along or with other components of the mitochondrial F0F1 ATPase (e.g.,F1 subunit). In particular, FIG. 11 shows a comparison of ATP hydrolysisbetween unsolvated Bz-423 and solvated Bz-423, FIG. 12 shows acomparison of ATP synthesis between unsolvated Bz-423 and solvatedBz-423, and FIG. 13 shows a comparison of cell viability betweenunsolvated Bz-423 and solvated Bz-423.

Example 4

This example demonstrates that unsolvated Bz-423 is more soluble insimulated gastric fluid than solvated Bz-423. Simulated gastric fluidsolubility at 37° C. analyses were conducted for solvated Bz-423, andanhydrous Bz-423. Anhydrous Bz-423 was more soluble than solvatedBz-423. FIG. 14 shows a UV-vis spectrum of Bz-423 in simulated gastricfluid. FIG. 15 shows a UV-vis spectrum of Bz-423 in simulated gastricfluid before and after addition of K₂CO₃.

Example 5

This example describes the formation of benzodiazepine formulations.FIG. 16 shows Raman spectroscopy data for Bz-423 ethanol solvate. BZ-423(12.3 mg) was dissolved completely in 0.5 mL of ethanol at 70° C. Uponcooling to room temperature crystallization of the solvate occurred. TheRaman spectrum above was obtained from the crystals. The characteristicRaman shifts for the ethanol solvate occur at about 1673 and 1560 cm⁻¹.

FIG. 17 shows Raman spectroscopy data for Bz-423 1-propanol solvate.BZ-423 (46.6 mg) was dissolved completely in 0.5 mL of 1-propanol at 70°C. Upon cooling to room temperature crystallization of the solvateoccurred. The Raman spectrum above was obtained from the crystals. Thecharacteristic Raman shifts for the 1-propanol solvate occur at about1671 and 1561 cm⁻¹.

FIG. 18 shows Raman spectroscopy data for Bz-423 2-propanol solvate.BZ-423 (53.2 mg) was dissolved completely in 0.5 mL of 2-propanol at 70°C. Upon cooling to room temperature crystallization of the solvateoccurred. The Raman spectrum above was obtained from the crystals. Thecharacteristic Raman shifts for the 2-propanol solvate occur at about1667 and 1562 cm⁻¹.

FIG. 19 shows Raman spectroscopy data for Bz-423 1-butanol solvate.BZ-423 (105.8 mg) was dissolved completely in 0.5 mL of 1-butanol at 70°C. Upon cooling to room temperature crystallization of the solvateoccurred. The Raman spectrum above was obtained from the crystals. Thecharacteristic Raman shifts for the 1-butanol solvate occur at about1661 and 1556 cm⁻¹.

FIG. 20 shows Raman spectroscopy data for Bz-423 2-butanol solvate.BZ-423 (97.3 mg) was dissolved completely in 0.5 mL of 2-butanol at 70°C. Upon cooling to room temperature crystallization of the solvateoccurred. The Raman spectrum above was obtained from the crystals. Thecharacteristic Raman shifts for the 2-butanol solvate occur at about1666 and 1562 cm⁻¹.

FIG. 21 shows Raman spectroscopy data for Bz-423 1-pentanol solvate.BZ-423 (99.3 mg) was dissolved completely in 0.5 mL of 1-pentanol at 70°C. Upon cooling to room temperature crystallization of the solvateoccurred. The Raman spectrum above was obtained from the crystals. Thecharacteristic Raman shifts for the 1-pentanol solvate occur at about1646 and 1563 cm⁻¹.

FIG. 22 shows Raman spectroscopy data for Bz-423 1-octanol solvate.BZ-423 (5.2 mg) was dissolved completely in 0.5 mL of 1-octanol at 70°C. Upon cooling to room temperature crystallization of the solvateoccurred. The Raman spectrum above was obtained from the crystals. Thecharacteristic Raman shifts for the 1-octanol solvate occur at about1669 and 1559 cm⁻¹.

FIG. 23 shows Raman spectroscopy data for Bz-423 propylene glycolsolvate. BZ-423 (16.8 mg) was dissolved completely in 0.5 mL ofpropylene glycol at 70° C. Upon cooling to room temperaturecrystallization of the solvate occurred. The Raman spectrum above wasobtained from the crystals. The characteristic Raman shifts for thepropylene glycol solvate occur at about 1660 and 1556 cm⁻¹.

FIG. 24 shows Raman spectroscopy data for Bz-423 acetone glass. BZ-423(14.3 mg) was dissolved completely in 0.5 mL of acetone at roomtemperature. Upon evaporation a glass was formed. The Raman spectrumabove was obtained from the glass. The characteristic Raman shifts forthe acetone glass occur at about 1674 and 1559 cm⁻¹.

All publications and patents mentioned in the above specification areherein incorporated by reference. Although the invention has beendescribed in connection with specific preferred embodiments, it shouldbe understood that the invention as claimed should not be unduly limitedto such specific embodiments. Indeed, various modifications of thedescribed modes for carrying out the invention that are obvious to thoseskilled in the relevant fields are intended to be within the scope ofthe following claims.

1. A composition comprising an unsolvated compound having the structuredescribed by the following formula:

including both R and S enantiomeric foms and racemic mixtures; whereinR1, R2, R3 and R4 are selected from the group consisting of: hydrogen;CH₃; a linear or branched, saturated or unsaturated aliphatic chainhaving at least 1 carbon; a linear or branched, saturated or unsaturatedaliphatic chain having at least 2 carbons, and having at least onehydroxy subgroup; a linear or branched, saturated or unsaturatedaliphatic chain having at least 2 carbons, and having at least one thiolsubgroup; a linear or branched, saturated or unsaturated aliphatic chainhaving at least 2 carbons, wherein said aliphatic chain terminates withan aldehyde subgroup; a linear or branched, saturated or unsaturatedaliphatic chain having at least 2 carbons, and having at least oneketone subgroup; a linear or branched, saturated or unsaturatedaliphatic chain having at least 2 carbons; wherein said aliphatic chainterminates with a carboxylic acid subgroup; a linear or branched,saturated or unsaturated aliphatic chain having at least 2 carbons, andhaving at least one amide subgroup; a linear or branched, saturated orunsaturated aliphatic chain having at least 2 carbons, and having atleast one acyl group; a linear or branched, saturated or unsaturatedaliphatic chain having at least 2 carbons, and having at least onenitrogen containing moiety; a linear or branched, saturated orunsaturated aliphatic chain having at least 2 carbons, and having atleast one amine subgroup; a linear or branched, saturated or unsaturatedaliphatic chain having at least 2 carbons, and having at least one ethersubgroup; a linear or branched, saturated or unsaturated aliphatic chainhaving at least 2 carbons, and having at least one halogen subgroup; alinear or branched, saturated or unsaturated aliphatic chain having atleast 2 carbons, and having at least one nitronium subgroup; wherein R5is selected from the group consisting of: OH; NO₂; OR′; wherein R′ isselected from the group consisting of: a linear or branched, saturatedor unsaturated aliphatic chain having at least one carbon; a linear orbranched, saturated or unsaturated aliphatic chain having at least 2carbons, and having at least one hydroxyl subgroup; a linear orbranched, saturated or unsaturated aliphatic chain having at least 2carbons, and having at least one thiol subgroup; a linear or branched,saturated or unsaturated aliphatic chain having at least 2 carbons,wherein said aliphatic chain terminates with an aldehyde subgroup; alinear or branched, saturated or unsaturated aliphatic chain having atleast 2 carbons, and having at least one ketone subgroup; a linear orbranched, saturated or unsaturated aliphatic chain having at least 2carbons; wherein said aliphatic chain terminates with a carboxylic acidsubgroup; a linear or branched, saturated or unsaturated aliphatic chainhaving at least 2 carbons, and having at least one amide subgroup; alinear or branched, saturated or unsaturated aliphatic chain having atleast 2 carbons, and having at least one acyl group; a linear orbranched, saturated or unsaturated aliphatic chain having at least 2carbons, and having at least one nitrogen containing moiety; a linear orbranched, saturated or unsaturated aliphatic chain having at least 2carbons, and having at least one amine subgroup; a linear or branched,saturated or unsaturated aliphatic chain having at least 2 carbons, andhaving at least one halogen subgroup; a linear or branched, saturated orunsaturated aliphatic chain having at least 2 carbons, and having atleast one nitronium subgroup; wherein R6 is selected from the groupconsisting of: Hydrogen; NO₂; Cl; F; Br; I; SR′; and NR′₂; wherein R′ isdefined as above in R5; wherein R7 is selected from the group consistingof: Hydrogen; a linear or branched, saturated or unsaturated aliphaticchain having at least 2 carbons; and wherein R8 is an aliphatic cyclicgroup larger than benzene; wherein said larger than benzene comprisesany chemical group containing 7 or more non-hydrogen atoms.
 2. Thecomposition of claim 1, wherein said compound is:


3. The composition of claim 1, wherein said unsolvated compound isanhydrous.
 4. The composition of claim 1, wherein said unsolvatedcompound has an orthorhombic crystal structure.
 5. A compositioncomprising a compound selected from the group consisting of Bz-423ethanol solvate, Bz-423 succinic acid, Bz-423 citric acid, BZ-423-aceticacid, BZ-423-CH₃CN, BZ-423-methanol, BZ-423-ethyl acetate,BZ-423-toluene, BZ-423-oxalic acid, BZ-423-fumaric acid, BZ-423-octanol,BZ-423-heptanoic acid, BZ-423-diphenyl ether, Bz-423 1-propanol solvate,Bz-423 2-propanol solvate, Bz-423 1-butanol solvate, Bz-423 2-butanolsolvate, Bz-423 1-pentanol solvate, Bz-423 propylene glycol, Bz-4231-octanol solvate, Bz-423 acetone glass, and BZ-423-trichlorobenzene. 6.A composition comprising an orthorhombic benzodiazepine crystal, saidbenzodiazepine having the structure:

including both R and S enantiomeric foms and racemic mixtures; whereinR1, R2, R3 and R4 are selected from the group consisting of: hydrogen;CH₃; a linear or branched, saturated or unsaturated aliphatic chainhaving at least 1 carbon; a linear or branched, saturated or unsaturatedaliphatic chain having at least 2 carbons, and having at least onehydroxy subgroup; a linear or branched, saturated or unsaturatedaliphatic chain having at least 2 carbons, and having at least one thiolsubgroup; a linear or branched, saturated or unsaturated aliphatic chainhaving at least 2 carbons, wherein said aliphatic chain terminates withan aldehyde subgroup; a linear or branched, saturated or unsaturatedaliphatic chain having at least 2 carbons, and having at least oneketone subgroup; a linear or branched, saturated or unsaturatedaliphatic chain having at least 2 carbons; wherein said aliphatic chainterminates with a carboxylic acid subgroup; a linear or branched,saturated or unsaturated aliphatic chain having at least 2 carbons, andhaving at least one amide subgroup; a linear or branched, saturated orunsaturated aliphatic chain having at least 2 carbons, and having atleast one acyl group; a linear or branched, saturated or unsaturatedaliphatic chain having at least 2 carbons, and having at least onenitrogen containing moiety; a linear or branched, saturated orunsaturated aliphatic chain having at least 2 carbons, and having atleast one amine subgroup; a linear or branched, saturated or unsaturatedaliphatic chain having at least 2 carbons, and having at least one ethersubgroup; a linear or branched, saturated or unsaturated aliphatic chainhaving at least 2 carbons, and having at least one halogen subgroup; alinear or branched, saturated or unsaturated aliphatic chain having atleast 2 carbons, and having at least one nitronium subgroup; wherein R5is selected from the group consisting of: OH; NO₂; OR′; wherein R′ isselected from the group consisting of: a linear or branched, saturatedor unsaturated aliphatic chain having at least one carbon; a linear orbranched, saturated or unsaturated aliphatic chain having at least 2carbons, and having at least one hydroxyl subgroup; a linear orbranched, saturated or unsaturated aliphatic chain having at least 2carbons, and having at least one thiol subgroup; a linear or branched,saturated or unsaturated aliphatic chain having at least 2 carbons,wherein said aliphatic chain terminates with an aldehyde subgroup; alinear or branched, saturated or unsaturated aliphatic chain having atleast 2 carbons, and having at least one ketone subgroup; a linear orbranched, saturated or unsaturated aliphatic chain having at least 2carbons; wherein said aliphatic chain terminates with a carboxylic acidsubgroup; a linear or branched, saturated or unsaturated aliphatic chainhaving at least 2 carbons, and having at least one amide subgroup; alinear or branched, saturated or unsaturated aliphatic chain having atleast 2 carbons, and having at least one acyl group; a linear orbranched, saturated or unsaturated aliphatic chain having at least 2carbons, and having at least one nitrogen containing moiety; a linear orbranched, saturated or unsaturated aliphatic chain having at least 2carbons, and having at least one amine subgroup; a linear or branched,saturated or unsaturated aliphatic chain having at least 2 carbons, andhaving at least one halogen subgroup; a linear or branched, saturated orunsaturated aliphatic chain having at least 2 carbons, and having atleast one nitronium subgroup; wherein R6 is selected from the groupconsisting of: Hydrogen; NO₂; Cl; F; Br; I; SR′; and NR′₂; wherein R′ isdefined as above in R5; wherein R7 is selected from the group consistingof: Hydrogen; a linear or branched, saturated or unsaturated aliphaticchain having at least 2 carbons; and wherein R8 is an aliphatic cyclicgroup larger than benzene; wherein said larger than benzene comprisesany chemical group containing 7 or more non-hydrogen atoms.
 7. Thecomposition of claim 6, wherein said compound is:


8. The composition of claim 6, wherein said orthorhombic benzodiazepinecrystal is anhydrous.
 9. A composition comprising an oral dose of abenzodiazepine having the structure:

including both R and S enantiomeric toms and racemic mixtures; whereinR1, R2, R3 and R4 are selected from the group consisting of: hydrogen;CH₃; a linear or branched, saturated or unsaturated aliphatic chainhaving at least 1 carbon; a linear or branched, saturated or unsaturatedaliphatic chain having at least 2 carbons, and having at least onehydroxy subgroup; a linear or branched, saturated or unsaturatedaliphatic chain having at least 2 carbons, and having at least one thiolsubgroup; a linear or branched, saturated or unsaturated aliphatic chainhaving at least 2 carbons, wherein said aliphatic chain terminates withan aldehyde subgroup; a linear or branched, saturated or unsaturatedaliphatic chain having at least 2 carbons, and having at least oneketone subgroup; a linear or branched, saturated or unsaturatedaliphatic chain having at least 2 carbons; wherein said aliphatic chainterminates with a carboxylic acid subgroup; a linear or branched,saturated or unsaturated aliphatic chain having at least 2 carbons, andhaving at least one amide subgroup; a linear or branched, saturated orunsaturated aliphatic chain having at least 2 carbons, and having atleast one acyl group; a linear or branched, saturated or unsaturatedaliphatic chain having at least 2 carbons, and having at least onenitrogen containing moiety; a linear or branched, saturated orunsaturated aliphatic chain having at least 2 carbons, and having atleast one amine subgroup; a linear or branched, saturated or unsaturatedaliphatic chain having at least 2 carbons, and having at least one ethersubgroup; a linear or branched, saturated or unsaturated aliphatic chainhaving at least 2 carbons, and having at least one halogen subgroup; alinear or branched, saturated or unsaturated aliphatic chain having atleast 2 carbons, and having at least one nitronium subgroup; wherein R5is selected from the group consisting of: OH; NO₂; OR′; wherein R′ isselected from the group consisting of: a linear or branched, saturatedor unsaturated aliphatic chain having at least one carbon; a linear orbranched, saturated or unsaturated aliphatic chain having at least 2carbons, and having at least one hydroxyl subgroup; a linear orbranched, saturated or unsaturated aliphatic chain having at least 2carbons, and having at least one thiol subgroup; a linear or branched,saturated or unsaturated aliphatic chain having at least 2 carbons,wherein said aliphatic chain terminates with an aldehyde subgroup; alinear or branched, saturated or unsaturated aliphatic chain having atleast 2 carbons, and having at least one ketone subgroup; a linear orbranched, saturated or unsaturated aliphatic chain having at least 2carbons; wherein said aliphatic chain terminates with a carboxylic acidsubgroup; a linear or branched, saturated or unsaturated aliphatic chainhaving at least 2 carbons, and having at least one amide subgroup; alinear or branched, saturated or unsaturated aliphatic chain having atleast 2 carbons, and having at least one acyl group; a linear orbranched, saturated or 0unsaturated aliphatic chain having at least 2carbons, and having at least one nitrogen containing moiety; a linear orbranched, saturated or unsaturated aliphatic chain having at least 2carbons, and having at least one amine subgroup; a linear or branched,saturated or unsaturated aliphatic chain having at least 2 carbons, andhaving at least one halogen subgroup; a linear or branched, saturated orunsaturated aliphatic chain having at least 2 carbons, and having atleast one nitronium subgroup; wherein R6 is selected from the groupconsisting of: Hydrogen; NO₂; Cl; F; Br; I; SR′; and NR′₂; wherein R′ isdefined as above in R5; wherein R7 is selected from the group consistingof: Hydrogen; a linear or branched, saturated or unsaturated aliphaticchain having at least 2 carbons; and wherein R8 is an aliphatic cyclicgroup larger than benzene; wherein said larger than benzene comprisesany chemical group containing 7 or more non-hydrogen atoms.
 10. Thecomposition of claim 9, wherein said compound is:


11. The composition of claim 9, wherein said benzodiazepine compound isanhydrous.
 12. The composition of claim 9, wherein said benzodiazepinecompound has an orthorhombic crystal structure.
 13. A method of treatingan autoimmune disorder or hyperproliferative disorder comprisingadministering to a subject an effective amount of a compositioncomprising the composition of claim 1.